Oncolytic Herpes Simplex Virus Infected Cells

ABSTRACT

A monocyte, monocyte derived cell or macrophage infected with an oncolytic herpes simplex virus is disclosed together with uses of such infected cells in the treatment of diseases such as cancer.

FIELD OF THE INVENTION

The present invention relates to a monocyte, monocyte derived cell ormacrophage infected with an oncolytic herpes simplex virus, and uses ofsuch infected cells in the treatment of diseases such as cancer.

BACKGROUND TO THE INVENTION

Cancer is one of the greatest concerns worldwide, because of the highmortality rates, the economic and social burden associated with it andthe psychological issues faced by cancer survivors.

Resistance to treatments is generally acquired when tumor mass presentsareas which are not reached or affected by conventional therapies, i.e.chemotherapy or radiotherapy. These regions are located at the centre ofthe tumor bulk and are generally characterised by a highly hypoxicenvironment, meaning that the oxygen supply is insufficient for theappropriate respiration of cells and stroma (Shannon, A. M., D. J.Bouchier-Hayes, C. M. Condron and D. Toomey, 2003 Tumour hypoxia,chemotherapeutic resistance and hypoxia-related therapies. CancerTreatment Reviews 29: 297-307). The hypoxic condition, which is aninvariable characteristic of solid tumors, develops because the rate ofcell replication in tumors overcomes that of vessel formation:continuous demand of oxygen, thus, is detected by cellular oxygensensors which address the need by stimulating the angiogenic sprouting.In turn, angiogenesis leads to the generation of structurally disorderedblood vessels with improper distribution within the cancer mass (Kandel,J. J., D. J. Yamashiro and J. Kitajewski, 2011 Angiogenesis in TumourDevelopment and Metastasis, pp. 81-93 in Therapeutic Angiogenesis forVascular Diseases: Molecular Mechanisms and Targeted Clinical Approachesfor the Treatment of Angiogenic Disease, edited by M. Slevin.Springer-Verlag Berlin, Berlin): this causes the development of adysfunctional microvasculature that leads to the inadequate diffusionand perfusion of oxygen throughout the mass of tumor (Vaupel, P., O.Thews and M. Hoeckel, 2001 Treatment resistance of solid tumors—Role ofhypoxia and anemia. Medical Oncology 18: 243-259). Ultimately, thiscreates a feedback loop which further increases hypoxia.

An important feature of hypoxic areas of cancers is the marked presenceof immune cells, which infiltrate into the tumor mass since the veryearly stages of cancer onset (Di Caro, G., F. Marchesi, L. Laghi and F.Grizzi, 2013 Immune cells: plastic players along colorectal cancerprogression. Journal of Cellular and Molecular Medicine 17: 1088-1095).Among the most studied cell types are tumor-associated macrophages(TAMs). TAMs are a population of macrophages that mobilise andaccumulate in great number in the hypoxic central areas of solid tumors(Turner, L., C. Scotton, R. Negus and F. Balkwill, 1999 Hypoxia inhibitsmacrophage migration. European Journal of Immunology 29: 2280-2287;Lewis, J. S., R. J. Landers, J. C. E. Underwood, A. L. Harris and C. E.Lewis, 2000 Expression of vascular endothelial growth factor bymacrophages is up-regulated in poorly vascularized areas of breastcarcinomas. Journal of Pathology 192: 150-158; Gollapudi, K., C. Galet,T. Grogan, H. Zhang, J. W. Said et al., 2013 Association betweentumor-associated macrophage infiltration, high grade prostate cancer,and biochemical recurrence after radical prostatectomy. American Journalof Cancer Research 3: 523-529). TAMs are characterised by a specificphenotype, activated in response to micro-environmental signals such ascytokines, growth factors and hormones (Martinez, F. O., S. Gordon, M.Locati and A. Mantovani, 2006 Transcriptional profiling of the humanmonocyte-to-macrophage differentiation and polarization: New moleculesand patterns of gene expression. Journal of Immunology 177: 7303-7311),that are often referred to as M2 macrophages. While their counterparts,M1-polarised macrophages, are activated in response to inflammatorymolecules and are characterised by high anti-tumor andimmuno-stimulatory functions, the M2-skewed macrophages express markedpro-tumor activities, suppressing inflammatory processes and promotingmatrix remodelling, invasion, angiogenesis and survival (Sica, A., T.Schioppa, A. Mantovani and P. Allavena, 2006 Tumour-associatedmacrophages are a distinct M2 polarised population promoting tumourprogression: Potential targets of anti-cancer therapy. European Journalof Cancer 42: 717-727).

TAMs are known to be recruited from the blood circulation by chemotacticcytokines which are continuously released by tumor cells; for instance,MCP-1, VEGF and CSF-1 expression was found to be positively correlatedwith TAM accumulation in numerous human tumors (Graves, D. T., R.Barnhill, T. Galanopoulos and H. N. Antoniades, 1992 EXPRESSION OFMONOCYTE CHEMOTACTIC PROTEIN-1 IN HUMAN-MELANOMA INVIVO. AmericanJournal of Pathology 140: 9-14; Kacinski, B. M., 1995 CSF-1 AND ITSRECEPTOR IN OVARIAN, ENDOMETRIAL AND BREAST-CANCER. Annals of Medicine27: 79-85. Arenberg, D. A., M. P. Keane, B. DiGiovine, S. L. Kunkel, S.R. B. Strom et aL, 2000 Macrophage infiltration in human non-small-celllung cancer: the role of CC chemokines. Cancer Immunology Immunotherapy49: 63-70; Lewis, J. S., R. J. Landers, J. C. E. Underwood, A. L. Harrisand C. E. Lewis, 2000 Expression of vascular endothelial growth factorby macrophages is up-regulated in poorly vascularized areas of breastcarcinomas. Journal of Pathology 192: 150-158). However, their specificaccumulation into hypoxic regions of tumors is fostered by severalfeatures: the marked presence of necrotic cells (Lewis, J., R. J.Landers, R. D. Leek, K. Corke, A. L. Harris et al., 1997 Role ofmacrophages in tumour angiogenesis: Regulation by hypoxia. Journal ofPathology 182: A1-A1) and the release of a number of chemo-attractants,such as MCP-1 (Li, X., H. Kimura, K. Hirota, H. Sugimoto and H. Yoshida,2005 Hypoxia reduces constitutive and TNF-alpha-induced expression ofmonocyte chemoattractant protein-1 in human proximal renal tubularcells. Biochemical and Biophysical Research Communications 335:1026-1034), VEGF (Brown, L. F., B. Berse, R. W. Jackman, K. Tognazzi, A.J. Guidi et al., 1995 EXPRESSION OF VASCULAR-PERMEABILITY FACTOR(VASCULAR ENDOTHELIAL GROWTH-FACTOR) AND ITS RECEPTORS IN BREAST-CANCER.Human Pathology 26: 86-91) and endothelins (Grimshaw, M. J., S. Naylorand F. R. Balkwill, 2002 Endothelin-2 is a hypoxia-induced autocrinesurvival factor for breast tumor cells. Molecular Cancer Therapeutics 1:1273-1281). Once amassed into hypoxic areas, TAMs respond tooxygen-depleted conditions through an increase in production and releaseof several factors, such as growth factors, MMPs and CXCLs, which inturn affect angiogenesis, cellular growth, invasive capabilities andmetastasis: thus, TAMs ultimately promote tumor progression (Sica, A.,T. Schioppa, A. Mantovani and P. Allavena, 2006 Tumour-associatedmacrophages are a distinct M2 polarised population promoting tumourprogression: Potential targets of anti-cancer therapy. European Journalof Cancer 42: 717-727).

Given their pivotal role in triggering cancer progression, infiltrationof TAMs in tumors has been correlated with poor prognosis in themajority of solid cancers: lung cancer (Chen, J. J. W., Y. C. Lin, P. L.Yao, A. Yuan, H. Y. Chen et al., 2005 Tumor-associated macrophages: Thedouble-edged sword in cancer progression. Journal of Clinical Oncology23: 953-964), oral squamous cell carcinoma (He, K.-F., L. Zhang, C.-F.Huang, S.-R. Ma, Y.-F. Wang et al., 2014 CD163+ Tumor-AssociatedMacrophages Correlated with Poor Prognosis and Cancer Stem Cells in OralSquamous Cell Carcinoma. BioMed research international 2014: 838632),papillary thyroid carcinoma (KIM et al. 2013), papillary renal cellcarcinoma (Behnes, C. L., F. Bremmer, B. Hemmerlein, A. Strauss, P.Strobel et al., 2014 Tumor-associated macrophages are involved in tumorprogression in papillary renal cell carcinoma. Virchows Archiv 464:191-196), breast cancer (Mukhtar, R. A., A. P. Moore, V. J. Tandon, O.Nseyo, P. Twomey et al., 2012 Elevated Levels of Proliferating andRecently Migrated Tumor-associated Macrophages Confer IncreasedAggressiveness and Worse Outcomes in Breast Cancer. Annals of SurgicalOncology 19: 3979-3986; Tang, X. Q., 2013 Tumor-associated macrophagesas potential diagnostic and prognostic biomarkers in breast cancer.Cancer Letters 332: 3-10), ovarian cancer (Lan, C. Y., X. Huang, S. X.Lin, H. Q. Huang, Q. C. Cai et al., 2013 Expression of M2-PolarizedMacrophages is Associated with Poor Prognosis for Advanced EpithelialOvarian Cancer. Technology in Cancer Research & Treatment 12: 259-267)and pancreatic cancer (Kurahara, H., S. Takao, T. Kuwahata, T. Nagai, Q.Ding et al., 2012 Clinical Significance of Folate Receptorbeta-expressing Tumor-associated Macrophages in Pancreatic Cancer.Annals of Surgical Oncology 19: 2264-2271).

Oncolytic virotherapy concerns the use of lytic viruses whichselectively infect and kill cancer cells. Some oncolytic viruses arepromising therapies as they display exquisite selection for replicationin cancer cells and their self-limiting propagation within tumorsresults in fewer toxic side effects. Several oncolytic viruses haveshown great promise in the clinic (Bell, J., Oncolytic Viruses: AnApproved Product on the Horizon? Mol Ther. 2010; 18(2): 233-234).

Macrophages are known to have a natural homing ability to a site ofdisease and have been proposed as cellular vehicles for gene therapy(Burke et al., Macrophages in gene therapy: cellular delivery vehiclesand in vivo targets. Journal of Leukocyte Biology Vol. 72, no.3417-428).

WO2007/113572 describes monocyte derived cells, e.g, macrophages, thatcomprise a magnetic material. The cells are described to be useful as avehicle for targeting a therapeutic agent to a diseased material in asubject, where the therapeutic agent may preferably be a gene (i.e. agene therapy approach to treatment of the diseased material) and thesubject requiring treatment is exposed to a magnetic field to assistlocation of the cells in the diseased material. Related work isdisclosed in Muthana et al. A novel magnetic approach to enhance theefficacy of cell-based gene therapies. Gene Therapy (2008) 15, 902-910.

Muthana et al., (Use of Macrophages to Target Therapeutic Adenovirus toHuman Prostate Tumors. Cancer Res; 71(5) Mar. 1, 2011) describe anapproach to treatment of prostate tumors in which macrophages weretransduced with a hypoxia-regulated E1A/B construct and an E1A-dependentoncolytic adenovirus, whose proliferation was also restricted toprostate tumor cells using prostate-specific promoter elements tocontrol E1A expression. In these experiments the macrophages were usedas ‘silent carriers’ of the adenovirus which was only induced toreplicate upon location in a hypoxic environment. Induction ofreplication of adenovirus did not lead to death of the macrophages. InMuthana et al., (Macrophage Delivery of an Oncoloytic Virus AbolishesTumor Regrowth and Metastasis after Chemotherapy or Irradiation. CancerRes; 73(2) Jan. 15, 2013) the authors describe experiments using thesame adenoviral approach to investigate the effects post-administrationof docetaxel or radiation therapy.

SUMMARY OF THE INVENTION

The present invention concerns a monocyte, monocyte derived cell ormacrophage which is infected with an oncolytic Herpes Simplex Virus. Theinfected monocyte, monocyte derived cell or macrophage is disclosed tobe useful in a method of treatment of disease, in particular thetreatment of cancer. Preferred treatments may include treatment of atissue or cancer that is hypoxic or a part of a tissue or cancer that ishypoxic. Preferred treatments may include treatment of a tissue orcancer located in deep tissues, organs or in the core of the body.

The infected cell represents a specialised vector which isself-targeting to diseased tissue, thereby delivering the oncolyticHerpes Simplex Virus directly to the diseased tissue, including tohypoxic regions of tissue which are otherwise very difficult topenetrate with therapeutic agents. The cells do not act merely as avector. Oncolytic Herpes Simplex Virus infection leads to death of themonocyte or monocyte derived cells, which may be caused by viralreplication and cell lysis. Cell death whilst present in the diseasedtissue therefore leads to release of oncolytic Herpes Simplex Virus anddirect delivery to the diseased cells, e.g. tumor cells, which can beinfected and lysed by oncolytic Herpes Simplex Virus.

Furthermore, the oncolytic Herpes Simplex Virus initiates an enhancedimmune response in hypoxic tissue, thereby promoting an immune responseto the disease, e.g. an anti-tumor immune response.

In some embodiments the monocyte, monocyte derived cell or macrophagemay also contain an exogenous magnetic material. In these embodimentsthe method of treatment may involve application of a magnetic field tothe subject in order to direct the monocyte, monocyte derived cell ormacrophage to a desired location in the subject's body where treatmentis required.

In one aspect of the present invention an ex vivo or in vitro monocyte,monocyte derived cell or macrophage infected with an oncolytic herpessimplex virus is provided.

In some embodiments the ex vivo or in vitro monocyte, monocyte derivedcell or macrophage may also contain an exogenous magnetic material.

In one aspect of the present invention a preparation comprising apopulation of monocytes, monocyte derived cells or macrophages infectedwith an oncolytic herpes simplex virus is provided.

In one embodiment the monocytes, monocyte derived cells or macrophagesalso contain an exogenous magnetic material.

In one aspect of the present invention, the preparation is provided foruse in a method of medical treatment, e.g. the treatment of cancer.

In another aspect of the present invention a method of preparing amonocyte, monocyte derived cell or macrophage infected with an oncolyticherpes simplex virus is provided, the method comprising contacting invitro a monocyte, monocyte derived cell or macrophage with an oncolyticherpes simplex virus.

In some embodiments the method further comprises contacting themonocyte, monocyte derived cell or macrophage with a magnetic material.

In another aspect of the present invention a method of producing apreparation comprising a population of monocytes, monocyte derived cellsor macrophages infected with an oncolytic herpes simplex virus isprovided, the method comprising providing a population of monocytes,monocyte derived cells or macrophages infected with an oncolytic herpessimplex virus, and formulating a preparation comprising said populationof cells.

In some embodiments, monocytes, monocyte derived cells or macrophages insaid population contain an exogenous magnetic material.

In another aspect of the present invention a monocyte, monocyte derivedcell or macrophage infected with an oncolytic herpes simplex virus, andoptionally containing an exogenous magnetic material, is provided foruse in a method of treatment of disease. The method of treatment mayoptionally comprise administering the monocyte, monocyte derived cell ormacrophage to a subject and applying a magnetic field to the subject inorder to direct cells containing an exogenous magnetic material to adesired location in the subject's body.

In another aspect of the present invention the use of a monocyte,monocyte derived cell or macrophage infected with an oncolytic herpessimplex virus, and optionally containing an exogenous magnetic material,in the manufacture of a medicament for use in the treatment of diseaseis provided. The treatment may optionally comprise administering themonocyte, monocyte derived cell or macrophage to a subject and applyinga magnetic field to the subject in order to direct cells containing anexogenous magnetic material to a desired location in the subject's body.

In another aspect of the present invention a preparation comprising apopulation of monocytes, monocyte derived cells or macrophages infectedwith an oncolytic herpes simplex virus, wherein the monocytes, monocytederived cells or macrophages contain an exogenous magnetic material, isprovided for use in a method of treatment of disease, the methodcomprising administering the preparation to a subject and applying amagnetic field to the subject in order to direct cells of theadministered preparation to a desired location in the subject's body.

In another aspect of the present invention the use of a population ofmonocytes, monocyte derived cells or macrophages infected with anoncolytic herpes simplex virus, wherein the monocytes, monocyte derivedcells or macrophages contain an exogenous magnetic material, in themanufacture of a medicament for use in a method of treatment of diseaseis provided, the method comprising administering the medicament to asubject and applying a magnetic field to the subject in order to directcells of the administered medicament to a desired location in thesubject's body.

In another aspect of the present invention a method of treating adisease in a subject in need of treatment is provided, the methodcomprising administering a preparation comprising a population ofmonocytes, monocyte derived cells or macrophages infected with anoncolytic herpes simplex virus to said subject, thereby treating saiddisease. Optionally, the monocytes, monocyte derived cells ormacrophages in said population may contain an exogenous magneticmaterial and the method may optionally further comprise applying amagnetic field to the subject in order to direct cells of theadministered preparation to a desired location in the subject's body.

Administration of cells infected with oncolytic Herpes Simplex Virus toa subject may be carried out within a predetermined time from infectionwith oncolytic Herpes Simplex Virus, and/or the administered cells maybe selected to contain a defined percentage range of dead or dying (e.g.lysed) cells, as described herein. The time and/or selection of cellsmay be such as to ensure that cells are undergoing cell death (e.g.undergoing lysis by the infected oncolytic Herpes Simplex Virus) whenthey are located in the tissue requiring treatment.

In another aspect of the present invention a kit of parts is provided,the kit comprising a predetermined amount of oncolytic Herpes SimplexVirus and a predetermined amount of a magnetic material. The kit may beprovided together with instructions for the infection of monocytes,monocyte derived cells or macrophages with the oncolytic Herpes SimplexVirus and/or for the loading of monocytes, monocyte derived cells ormacrophages with the magnetic material. Such instructions may be forcarrying out said infection and/or loading ex vivo or in vitro, e.g.under conditions of in vitro cell culture.

In some embodiments all copies of the ICP34.5 gene in the genome of theoncolytic herpes simplex virus are modified such that the ICP34.5 geneis incapable of expressing a functional ICP34.5 gene product. As suchthe oncolytic herpes simplex virus may be an ICP34.5 null mutant.

In some embodiments one or both of the ICP34.5 genes in the genome ofthe oncolytic herpes simplex virus are modified such that the ICP34.5gene is incapable of expressing a functional ICP34.5 gene product.

In some embodiments the oncolytic herpes simplex virus is a mutant ofHSV-1 strain 17. In preferred embodiments the oncolytic herpes simplexvirus is HSV1716 (ECACC Accession No. V92012803). In some embodimentsthe herpes simplex virus is a mutant of HSV-1 strain 17 mutant 1716.

In some embodiments the disease to be treated is a cancer, e.g. a tumor.The treatment may be of a hypoxic region of the cancer, which may alsobe the desired location to which cells are magnetically directed. Assuch, methods of treatment may involve treatment of a hypoxic region ofa cancer, either together with other normoxic regions of the cancer orindependent of treatment of normoxic regions of the cancer. The methodof treatment may also involve induction of an anti-tumor response in thesubject. The method of treatment may also involve applying a magneticfield to the subject in order to direct administered cells containing anexogenous magnetic material to a hypoxic region of the cancer.

Description

The inventors' findings indicate that an oncolytic Herpes Simplex Virusis able to kill monocyte derived cells after 96 hours from infection.Oncolytic Herpes Simplex Virus are generally known to have a highlyselective lytic activity towards proliferating cells, in principlepermitting tumor treatment by systemic or non-localised administrationand self-targeting of tumor cells without harming healthy cells. Thisaffords an exemplary safety profile.

The inventors' have observed that following infection of monocytederived cells virus is not detected in the infected cells but presenceof virus is re-established upon prolonged culture (FIG. 1). This isconsistent with productive infection of the cells, i.e. involvingreplication and cell lysis by viral progeny, although the invention isnot bound by such theory. The finding that infection with oncolyticHerpes Simplex Virus leads to cell death of monocyte derived cells meansthat infected monocytes or monocyte derived cells may be used to deliverthe oncolytic Herpes Simplex Virus to the diseased tissue, including tohypoxic areas of a tumor, subsequently allowing the release of virusdirectly to the diseased tissue as the cell dies.

The inventors also found that oncolytic Herpes Simplex Virus replicationin, and subsequent cell death (e.g. lysis) of, monocyte derived cells isactually greater in hypoxic conditions. This indicates that death of themonocyte derived cells occurs (apparently preferentially) in hypoxictumor environments and will directly release the oncolytic HerpesSimplex Virus to the hypoxic parts of a tumor that are otherwisedifficult to access.

Accordingly, to ensure that a substantial number of monocyte or monocytederived cells will undergo death and viral release when they are presentin the target tissue or tumor, administration of infected cells to asubject may be carried out within a predetermined time from infectionwith oncolytic Herpes Simplex Virus, and/or the administered cells maybe selected to contain a defined percentage range of dying or dead (e.g.lysed) cells. The inventors have also shown that monocyte or monocytederived cells infected with oncolytic Herpes Simplex Virus and loadedwith an exogenous magnetic material can be steered from the bloodstreaminto deep tissue targets (including primary and secondary (metastatic)tumors) by magnetic resonance targeting. By way of example, this may usepulsed magnetic-field gradients within a magnetic resonance (e.g. MRI,MRT) system. Accordingly, systemic administration, e.g. to the blood,coupled with accurate non-invasive direction of the oncolytic HerpesSimplex Virus armed cells directly to the diseased tissue becomes areality.

This approach has particular application when a tissue or tumor isdifficult or impossible to remove surgically, e.g. as in the lung,brain, liver or spinal cord. Additionally, targeting of cells to one ormore metastatic lesions in cancer patients is possible using phasedadministration of cells, coupled with respective independent magneticresonance sessions to target each administration of cells to independentlocations.

In addition to direct action of oncolytic herpes simplex virus (oHSV) ontumors, there is growing evidence that the host immune response plays animportant role in establishing the efficacy of the anti-tumor responsethrough innate immune effectors, adaptive antiviral immune responses andadaptive antitumor immune responses (e.g. see Prestwich et al.,Onoclytic viruses: a novel form of immunotherapy. Expert Rev AnticancerTher. October 2008; 8(10): 1581-1588).

Several studies have shown that oHSV is capable of inducing ananti-tumor immune response. This can manifest as tumor growth reductionin lesions treated with oHSV and in untreated lesions in the sameanimal, efficacy of oHSV requiring an intact immune response, inductionof anti-tumor cytokine response, reversal of tumor immune dysfunctionand facilitation of tumor antigen presentation. Induction of ananti-tumor immune response can reduce establishment of metastases, orcontribute to their elimination, and protect from re-occurrence oftumor.

For example, in Benencia et al., ((2008) Herpes virus oncolytic therapyreverses tumor immune dysfunction and facilitates tumor antigenpresentation. Cancer Biol.Ther. 7, 1194-1205) growth reduction intreated and untreated lesions was reported. In Miller and Fraser ((2003)Requirement of an integrated immune response for successfulneuroattenuated HSV-1 therapy in an intracranial metastatic melanomamodel. Mol. Ther. 7(6):741-747) efficacy of HSV176 required an intactimmune response which was mediated by a tumor-specific proliferative Tcell response.

The inventors have shown here that in hypoxic conditions oncolyticHerpes Simplex Virus is an inducer of pro-inflammatory cytokines andtranscription factors (e.g., IL-8, IL-1 and NFκB), relative to normoxicconditions. These findings suggest increased inflammatory responseproperties of oncolytic Herpes Simplex Virus in hypoxic conditions,suggesting that oncolytic Herpes Simplex Virus acquires a greater viralpotentiality in hypoxia, further supporting the rationale of using virusdelivery by monocytes or monocyte derived cells to target central areasof a tumor that are hypoxic and difficult to access.

Oncolytic Herpes Simplex Virus

An oncolytic virus is a virus that will lyse cancer cells (oncolysis),preferably in a preferential or selective manner. Viruses thatselectively replicate in dividing cells over non-dividing cells areoften oncolytic. Oncolytic viruses are well known in the art and arereviewed in Molecular Therapy Vol. 18 No. 2 February 2010 pg 233-234.

The herpes simplex virus (HSV) genome comprises two covalently linkedsegments, designated long (L) and short (S). Each segment contains aunique sequence flanked by a pair of inverted terminal repeat sequences.The long repeat (RL or R_(L)) and the short repeat (RS or R_(S)) aredistinct.

The HSV ICP34.5 (also called y34.5) gene, which has been extensivelystudied, has been sequenced in HSV-1 strains F and syn17+ and in HSV-2strain HG52. One copy of the ICP34.5 gene is located within each of theRL repeat regions. Mutants inactivating one or both copies of theICP34.5 gene are known to lack neurovirulence, i.e. beavirulent/non-neurovirulent (non-neurovirulence is defined by theability to introduce a high titre of virus (approx 10⁶ plaque formingunits (pfu)) to an animal or patient without causing a lethalencephalitis such that the LD₅₀ in animals, e.g. mice, or human patientsis in the approximate range of ≥10⁶ pfu), and be oncolytic.

Preferred oncolytic Herpes Simplex Virus (oHSV) arereplication-competent virus, being replication-competent at least in thetarget tumor/cancer cells.

Oncolytic HSV that may be used in the present invention include HSV inwhich one or both of the γ34.5 (also called ICP34.5) genes are modified(e.g. by mutation which may be a deletion, insertion, addition orsubstitution) such that the respective gene is incapable of expressing,e.g. encoding, a functional ICP34.5 protein. Preferably, in HSVaccording to the invention both copies of the γ34.5 gene are modifiedsuch that the modified HSV is not capable of expressing, e.g. producing,a functional ICP34.5 protein.

In some embodiments the oncolytic herpes simplex virus may be an ICP34.5null mutant where all copies of the ICP34.5 gene present in the herpessimplex virus genome (two copies are normally present) are disruptedsuch that the herpes simplex virus is incapable of producing afunctional ICP34.5 gene product. In other embodiments the oncolyticherpes simplex virus may lack at least one expressible ICP34.5 gene. Insome embodiments the herpes simplex virus may lack only one expressibleICP34.5 gene. In other embodiments the herpes simplex virus may lackboth expressible ICP34.5 genes. In still other embodiments each ICP34.5gene present in the herpes simplex virus may not be expressible. Lack ofan expressible ICP34.5 gene means, for example, that expression of theICP34.5 gene does not result in a functional ICP34.5 gene product.

Oncolytic herpes simplex virus may be derived from any HSV including anylaboratory strain or clinical isolate (non-laboratory strain) of HSV. Insome preferred embodiments the HSV is a mutant of HSV-1 or HSV-2.Alternatively the HSV may be an intertypic recombinant of HSV-1 andHSV-2. The mutant may be of one of laboratory strains HSV-1 strain 17,HSV-1 strain F or HSV-2 strain HG52. The mutant may be of thenon-laboratory strain JS-1. Preferably the mutant is a mutant of HSV-1strain 17. The herpes simplex virus may be one of HSV-1 strain 17 mutant1716, HSV-1 strain F mutant R3616, HSV-1 strain F mutant G207, HSV-1mutant NV1020, or a further mutant thereof in which the HSV genomecontains additional mutations and/or one or more heterologous nucleotidesequences. Additional mutations may include disabling mutations, whichmay affect the virulence of the virus or its ability to replicate. Forexample, mutations may be made in any one or more of ICP6, ICP0, ICP4,ICP27. Preferably, a mutation in one of these genes (optionally in bothcopies of the gene where appropriate) leads to an inability (orreduction of the ability) of the HSV to express the correspondingfunctional polypeptide. By way of example, the additional mutation ofthe HSV genome may be accomplished by addition, deletion, insertion orsubstitution of nucleotides.

A number of oncolytic herpes simplex viruses are known in the art.Examples include HSV1716, 83616 (e.g. see Chou & Roizman, Proc. Natl.Acad. Sci. Vol.89, pp.3266-3270, April 1992), G207 (Toda et al, HumanGene Therapy 9:2177-2185, Oct. 10, 1995), NV1020 (Geevarghese et al,Human Gene Therapy 2010 Sep; 21(9):1119-28), RE6 (Thompson et al,Virology 131, 171-179 (1983)), and Oncovex™ (Simpson et al, Cancer Res2006; 66:(9) 4835-4842 May 1, 2006; Liu et al, Gene Therapy (2003): 10,292-303), dlsptk, hrR3,R4009, MGH-1, MGH-2, G47Δ, Myb34.5, DF3γ34.5,HF10, NV1042, RAMBO, rQNestin34.5, R5111, R-LM113, CEAICP4, CEAγ34.5,DF3γ34.5, KeM34.5 (Manservigi et al, The Open Virology Journal 2010;4:123-156), rRp450, M032 (Campadelli-Fiume et al, Rev Med. Virol 2011;21:213-226), Baco1 (Fu et al, Int. J. Cancer 2011; 129(6):1503-10) andM032 and C134 (Cassady et al, The Open Virology Journal 2010;4:103-108).

In some preferred embodiments the herpes simplex virus is HSV-1 strain17 mutant 1716 (HSV1716). HSV 1716 is an oncolytic, non-neurovirulentHSV and is described in EP 0571410, WO 92/13943, Brown et al (Journal ofGeneral Virology (1994), 75, 2367-2377) and MacLean et al (Journal ofGeneral Virology (1991), 72, 631-639). HSV 1716 has been deposited on 28January 1992 at the European Collection of Animal Cell Cultures, VaccineResearch and Production Laboratories, Public Health Laboratory Services,Porton Down, Salisbury, Wiltshire, SP4 OJG, United Kingdom underaccession number V92012803 in accordance with the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure (herein referred toas the ‘Budapest Treaty’).

In some embodiments the herpes simplex virus is a mutant of HSV-1 strain17 modified such that both ICP34.5 genes do not express a functionalgene product, e.g. by mutation (e.g. insertion, deletion, addition,substitution) of the ICP34.5 gene, but otherwise resembling orsubstantially resembling the genome of the wild type parent virus HSV-1strain 17+. That is, the virus may be a variant of HSV1716, having agenome mutated so as to inactivate both copies of the ICP34.5 gene ofHSV-1 strain 17+ but not otherwise altered to insert or delete/modifyother protein coding sequences.

In some embodiments the genome of an oncolytic Herpes Simplex Virusaccording to the present invention may be further modified to containnucleic acid encoding at least one copy of a polypeptide that isheterologous to the virus (i.e. is not normally found in wild typevirus) such that the polypeptide can be expressed from the nucleic acid.As such, the oncolytic virus may also be an expression vector from whichthe polypeptide may be expressed. Examples of such viruses are describedin WO2005/049846 and WO2005/049845.

In order to effect expression of the polypeptide, nucleic acid encodingthe polypeptide is preferably operably linked to a regulatory sequence,e.g. a promoter, capable of effecting transcription of the nucleic acidencoding the polypeptide. A regulatory sequence (e.g. promoter) that isoperably linked to a nucleotide sequence may be located adjacent to thatsequence or in close proximity such that the regulatory sequence caneffect and/or control expression of a product of the nucleotidesequence. The encoded product of the nucleotide sequence may thereforebe expressible from that regulatory sequence.

In some preferred embodiments, the oncolytic Herpes Simplex Virus is notmodified to contain nucleic acid encoding at least one copy of apolypeptide (or other nucleic acid encoded product) that is heterologousto the virus. That is the virus is not an expression vector from which aheterologous polypeptide or other nucleic acid encoded product may beexpressed. Such oHSV are not suitable for, or useful in, gene therapymethods and the method of medical treatment for which they are employedmay optionally be one that does not involve gene therapy.

Monocyte, Monocyte Derived Cell or Macrophage

Monocytes are a type of leukocyte (white blood cell) produced by thebone marrow. Following initial circulation in the blood they normallymove into tissues where they differentiate into macrophages or dendriticcells. Monocytes and their macrophage and dendritic cell progeny areinvolved in phagocytosis, antigen presentation and cytokine production.

Phagocytosis involves the uptake (ingestion) of matter (e.g. microbialor particulate matter or, in some instances, of nutrients) into the cellfollowed by digestion and/or destruction of the matter within the cell.Phagocytosis is a specialised form of endocytosis. The process ofphagocytosis usually involves engulfing the matter in a membrane boundvesicle (the phagosome) which is internalised into the cell. Thephagosome may then fuse with a lysosome to form a phagolysosome in whichdigestion of the matter may occur. Considering the role of monocytes andtheir progeny in the innate immune system, phagocytosis plays a majorrole in the removal of pathogens and cell debris.

Monocytes or monocyte derived cells may be isolated from peripheralblood or other tissues (e.g. see de Almeida et al (A Simple Method forHuman Peripheral Blood Monocyte Isolation. Mem Inst Oswaldo Cruz, Rio deJaneiro, Vol. 95(2): 221-223, March/April 2000); Elkord et al (Humanmonocyte isolation methods influence cytokine production from in vitrogenerated dendritic cells. Immunology. Feb 2005; 114(2):204-212); Repniket al (Simple and cost-effective isolation of monocytes from buffycoats. Journal of Immunological Methods Vol. 278, Issues 1-2, July 2003,pages 283-292); Zhang et al (The Isolation and Characterization ofMurine Macrophages. Curr. Protoc. Immunol. 83:14.1.1-14.1.14. 2008);John Q. Davies and Siamon Gordon (The Isolation and Culture of HumanMacrophages. Basic Cell Culture Protocols Methods in Molecular BiologyVol 290, 2005, pp105-116).

Macrophages are mononuclear phagocytes that are widely distributedthroughout the body, where they participate in innate and adaptiveimmune responses. The physiology of macrophages can vary depending onthe tissue environment in which they reside and the local stimuli towhich they are exposed. As such a range of different tissue-specificmacrophages can be identified, e.g. adipose tissue macrophages fromadipose tissue, monocytes from blood or bone marrow, Kupffer cells fromliver. Macrophages are secretory cells, and can promote and regulateimmune responses by secretion of cytokines and chemokines. Humanmacrophages can be isolated by flow cytometry in view of their specificexpression of proteins such as CD14, CD40, Cd11b and CD64. Macrophagescan be isolated from other mammals, e.g. mice or other rodents, bysimilar techniques. For reviews of monocytes and macrophages see NatureReviews Immunology, 11, (2011).

Monocytes and their progeny such as macrophages are attracted to hypoxictissue (tissue having a low oxygen tension), which is a hallmark featureof mammalian and experimental tumors, often because of limited tumorvascularisation. Monocytes are continually recruited into tumors wherethey accumulate and differentiate into tumor associated macrophages(TAMs). TAMs are abundant in solid and haemotological malignancies andhave been linked with progression, metastasis and resistance to therapy(Cook and Hagemann. Tumor-associated macrophages and cancer. CurrentOpinion in Pharmacology, Vol. 12, Issue 4, August 2013, pages 595-601).Studies have shown that macrophages respond to levels of hypoxia foundin tumors by up-regulating hypoxia inducible transcription factors whichactivate a broad range of mitogenic, proinvasive, proangiogenic andprometastatic genes (Lewis and Murdoch. Macrophage Responses to Hypoxia,Implications for Tumor progression and Anti-Cancer Therapies. Am JPathol. September 2005; 167(3): 627-635).

The present invention is concerned with monocytes, or monocyte derivedcells such as macrophages or dendritic cells capable of being infectedwith an oncolytic Herpes Simplex Virus, and optionally capable ofuptaking an exogenous magnetic material, e.g. by phagocytosis, toproduce a cell that is ‘loaded’ with the magnetic material. The cellsmay be non-human, preferably mammalian e.g. rabbit, guinea pig, rat,mouse or other rodent (including cells from any animal in the orderRodentia), cat, dog, pig, sheep, goat, cattle, horse, non-human primate,or may be human cells. In some embodiments it is preferred that the cellis a macrophage, e.g. a human or mammalian macrophage.

The monocyte or monocyte derived cell may be isolated or obtained from asubject to be treated, e.g. by isolation from a sample of peripheralblood as described above. Alternatively, it may be isolated or obtainedfrom a donor subject, e.g. another mammal or human (preferably of thesame species). Donor monocytes may be screened for immunocompatibility.Monocytes or monocyte derived cells may be isolated from other celltypes to provide a culture or preparation that is substantially free ofcells that are not monocytes or monocyte derived cells. Optionally,suitable support or feeder cells may be present in the culture orpreparation.

The isolated cells may be cultured in vitro where they may be infectedwith the oncolytic Herpes Simplex Virus and loaded with the magneticmaterial.

Infection of monocytes or monocyte derived cells refers to contactingthe cells with oncolytic Herpes Simplex Virus under conditions and for asufficient amount of time suitable to allow the Herpes Simplex Virus toenter the cells. Such infection may preferably be performed underconditions of in vitro cell culture. Techniques for the in vitroinfection of human and mammalian cells are known to those of ordinaryskill in the art, e.g. see Szántó et al. Peristent infection of BHKcells with herpes simplex virus types 1 and 2 in the absence of specificanti-herpetic antibody. Acta Virol. 1976 February; 20(1); 40-7); Conneret al. Herpes simplex virus type 1 strain HSV1716 grown in baby hamsterkidney cells has altered tropism for non-permissive Chinese hamsterovary cells compared to HSV1716 grown in vero cells. J Virol. 2005August; 79(15):9970-81. Techniques for the in vitro culture ofmacrophages are also known to those of ordinary skill in the art, e.g.see John Q. Davies and Siamon Gordon (The Isolation and Culture of HumanMacrophages. Basic Cell Culture Protocols Methods in Molecular BiologyVol. 290, 2005, pp105-116).

Accordingly, methods of preparing a monocyte, monocyte derived cell ormacrophage infected with an oncolytic herpes simplex virus, may comprisecontacting one or a plurality (optionally a population) of monocytes,monocyte derived cells or macrophages with a quantity of oncolyticherpes simplex virus under suitable conditions, e.g. of in vitro cellculture, and for sufficient time to permit productive infection of themonocytes, monocyte derived cells or macrophages.

Optionally, the cells may be maintained in culture under conditions inwhich the virus is able to induce cell death. Although not wishing to bebound by theory, the inventors believe that following infection of amonocyte, monocyte derived cell or macrophage oncolytic herpes simplexundergoes replication and viral progeny subsequently lyse the cells,causing cell death. As such, the culture conditions and duration may besuitable to produce a culture having a mixture of intact and dead, e.g.lysed, monocytes, monocyte derived cells or macrophages.

Cells may be contacted with a virus so as to achieve a multiplicity ofinfection (MOI) in the range 0.5-100, optionally one of 0.5-5, 1-10,10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 1-30,5-30, 5-50, or 30-50.

Optionally, cells may be administered to a subject within apredetermined time from infection. This may be to ensure that death(e.g. lysis) of cells occurs in the target tissue or tumor, allowingdissemination and spread of virus in the target tissue. As such,administration of cells may be within 1 hour, 2 hours, 3 hours, 6 hours,12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 3days, 4 days, 5 days, 6 days or 7 days, 8 days, 9 days, 10 days, 11days, 12 days, 13 days, or 14 days of infection.

Loading of monocytes or monocyte derived cells with magnetic materialmay also be performed under in vitro culture conditions and this stepmay be performed prior to, together with, or after infection of thecells with the oncolytic Herpes Simplex Virus. Techniques for loadingmonocytes or monocyte derived cells with magnetic material are, forexample, described in Kaim et al. MR imagaing with ultrasmallsuperparamagentic iron oxide particles in experimental soft-tissueinfections in rats. Radiology 2002 December; 225(3):808-14, and inMuthana et al. A novel magnetic approach to enhance the efficacy ofcell-based gene therapies. Gene Therapy (2008) 15, 902-910). Cells maybe loaded with magnetic material by contacting the cells with asuspension of magnetic particles having a concentration of particles ofabout 20 to 300 μ/ml, or one of about 50 to 150 μg/ml, 75 to 125 μg/ml,90 to 110 μ/ml or about 100 μ/ml.

Accordingly, methods of preparing a monocyte, monocyte derived cell ormacrophage may comprise, in addition to infection with an oncolyticherpes simplex virus, the step of contacting the cells with a quantityof magnetic material under suitable conditions, e.g. of in vitro cellculture, and for sufficient time to permit uptake, e.g. by phagocytosis,of magnetic material into monocytes, monocyte derived cells ormacrophages.

Following infection and/or uptake of magnetic material, cells may befurther cultured for as long as is desired, collected, isolated,purified or separated and formulated into a suitable preparation.

The monocytes, monocyte derived cells or macrophages described hereinmay be formulated as preparations, e.g. pharmaceutical compositions ormedicaments for clinical use and in such formulations may be combinedwith a pharmaceutically acceptable carrier, diluent or adjuvant. Amethod of formulating or producing a preparation may comprise mixing theselected cells, with a pharmaceutically acceptable carrier, adjuvant,diluent or buffer.

A preparation may comprise a population of monocytes, monocyte derivedcells or macrophages meaning that the preparation is made up of aplurality of said cells having common characteristics, e.g. monocyte,monocyte derived cells or macrophages infected with an oncolytic HerpesSimplex Virus, and optionally containing an exogenous magnetic material.The preparation may take any form, as described herein. Purely by way ofexample, the preparation may be a pharmaceutical composition ormedicament comprising the population of cells together with apharmaceutically acceptable carrier, adjuvant, diluent or buffer.

By way of example, the preparation may be formulated for parenteral,systemic, intracavitary, intravenous, intra-arterial or intratumoralroutes of administration which may include injection or delivery bycatheter. Suitable formulations may comprise the cells in a sterile orisotonic medium. Medicaments and pharmaceutical compositions may beformulated in fluid form suitable for injection, e.g. as a liquid,solution, suspension, or emulsion, or may be formulated as a depot orreservoir, e.g. suitable for implantation in the subject's body, fromwhich the rate of release of the cells may be controlled. Depotformulations may include gels, pastes, boluses or capsules. Thepreparation may be provided in a suitable container or packaging. Fluidformulations may be formulated for administration by injection or viacatheter to a selected region of the human or animal body.

The term “pharmaceutically acceptable” as used herein pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, adjuvant, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation. Suitable carriers, adjuvants, excipients, etc. can be foundin standard pharmaceutical texts, for example, Remington'sPharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton,Pa., 1990; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

A population of cells refers to a plurality of cells having commoncharacteristics, e.g. monocyte, monocyte derived cells or macrophagesinfected with an oncolytic Herpes Simplex Virus, and optionallycontaining an exogenous magnetic material. In some embodiments thepopulation will contain approximately several hundred cells or more of agiven type i.e. of monocytes, monocyte derived cells or macrophages. Thepopulation may have several thousand cells or more of the given type ora number of cells of the approximate order 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷ ormore. A population may exist in, or be isolated from, an in vitroculture of cells, or may exist in a preparation of cells, e.g. in apharmaceutical composition or medicament.

The population of cells may refer to all cells, or substantially allcells, in a culture or preparation being of the given type, i.e. allcells, or substantially all cells, in the culture or preparation beingmonocytes, monocyte derived cells or macrophages. In some embodiments apreparation or culture of cells may contain other types of cell, e.g.feeder cells or fibroblasts, which may optionally not be considered partof the population. In some embodiments it is preferred that in apopulation of cells at least 80% of the cells are monocytes, monocytederived cells or macrophages. In some embodiments this percentage may beone of 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or 100%.

Preferably, in a population of cells at least 80%, or one of 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%of the monocytes, monocyte derived cells or macrophages in thepopulation are infected with the oHSV, and optionally also contain anexogenous magnetic material. In some preferred embodiments substantiallyall, e.g. 95% or more, of the monocytes, monocyte derived cells ormacrophages in the population are infected with the oHSV and optionallyalso contain an exogenous magnetic material.

In a population of cells, some of the monocytes, monocyte derived cellsor macrophages that are infected with oHSV may have undergone cell death(e.g. lysed by the oHSV). Dying or dead (e.g. lysed) cells may accountfor 1-50% of the monocytes, monocyte derived cells or macrophages thatare infected with oHSV or of the population of monocytes, monocytederived cells or macrophages. In some embodiments this range may be oneof 0.5%-5%, 1-5%, 1-10%, 1-20%, 10-20%, 10-30%, 20-40% or 30-50%. Thismay be the percentage of dying or dead (e.g. lysed) cells in apopulation at the time of administration to a subject.

In some preferred embodiments, the monocyte, monocyte derived cell ormacrophage is not modified to contain nucleic acid encoding at least onecopy of a polypeptide (or other nucleic acid encoded product) that isheterologous to the cell. That is the cell is not modified to expressthe heterologous polypeptide or other nucleic acid encoded product. Suchcells are not suitable for, or useful in, gene therapy methods and themethod of medical treatment for which they are employed may optionallybe one that does not involve gene therapy (i.e. a medical method ortreatment reliant on expression of an heterologous polypeptide or othernucleic acid encoded product).

Optionally, and as described elsewhere herein, the oHSV with which themonocyte, monocyte derived cell or macrophage is infected may also beone that is not modified to contain a nucleic acid encoding apolypeptide (or other nucleic acid encoded product) that is heterologousto the virus and as such is also not suitable for, or useful in, genetherapy methods, and the method of medical treatment for which they areemployed may optionally be one that does not involve gene therapy.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to the individual. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of the disease being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

In some embodiments replication or proliferation of the oncolytic HerpesSimplex Virus is not responsive to an hypoxic environment. That is, theoncolytic Herpes Simplex Virus is not modified (or further modified),relative to the wild type virus or to a parental oncolytic virus (e.g.HSV1716), so as to become responsive to an hypoxic environment. Forexample, viral replication and/or gene expression is not under thecontrol of one or more regulatory elements, e.g. promoter(s), that areresponsive (e.g. activated or repressed) to hypoxia in the infected cellor surrounding tissue.

In some embodiments the oncolytic Herpes Simplex Virus is not modified(or further modified), relative to the wild type virus or to a parentaloncolytic virus (e.g. HSV1716), so as to replicate or proliferate inspecific tissue types, including tumor tissue. For example, viralreplication and/or gene expression is not under the control of one ormore regulatory elements, e.g. promoter(s), that respond (e.g. areactivated or repressed), to location in a specific tissue. For example,viral replication and/or gene expression is not placed under the controlof one or more tissue specific or tumor specific promoters (or otherregulatory elements).

Infection of Monocytes, Monocyte Derived Cells or Macrophages InducesFormation of a Distinct Population of Cells

Gene expression analysis of macrophages infected with oncolytic HerpesSimplex Virus indicates that following infection the cells undergo achange in expression of certain factors, including some pro-inflammatorycytokines, such as IL-6, IL-8, TNF-α, IL-1, CXCL-1, someanti-inflammatory cytokines, such as IL-10, CXCL-6 and other factors,such as NFκB, VEGF-A, and TGF-β.

As such, infection with oncolytic Herpes Simplex Virus leads toformation of a distinct population of cells characterised as beingmonocytes, monocyte derived cells or macrophages having a distinctpattern of expression of certain genes/proteins. The cells may furtherbe characterised by the conditions of culture or formulation, i.e.normoxic (about 18 to 22% pO₂) or hypoxic (less than 5% pO₂ andpreferably 0.1 to 3% pO₂). The cells may be provided as an in vitro orex vivo preparation of cells, optionally isolated or purified, and maybe cells maintained in culture or in a formulation under respectivenormoxic or hypoxic conditions.

In some embodiments, compared to uninfected cells of the same type,expression of one or more of the pro-inflammatory cytokines IL-6, IL-8,TNF-α, IL-1, CXCL-1 may be upregulated. Such upregulation may preferablyoccur when the cells are in hypoxic conditions (e.g. about 0.1% pO₂).Upregulation of IL-8 and/or IL-1 may be at least 2-fold, optionally3-fold, or 5-fold. Upregulation of NFκB or TGF-β or CXCL6 expression mayalso be observed under hypoxic conditions. Upregulation of NFκB andother factors may be consistent with induction of a type 1 T cellresponse (Th1 and/or Tc1), which is desirable for the treatment ofcancers and may be additive to an anti-virus Th1 type immune responseinitiated in the subject when viral particles are released from thecells.

In some embodiments, compared to uninfected cells of the same type,expression of one or more of IL-8, IL-1, NFκB, IL-10, and VEGFA may beupregulated. Such upregulation may preferably occur when the cells arein hypoxic conditions (e.g. less than 5% pO₂ and preferably 0.1 to 3%pO₂). Such upregulation may be at least 2-fold, optionally 3-fold, or5-fold, or more.

In some embodiments, compared to uninfected cells of the same type,expression of one or more of the anti-inflammatory cytokines IL-10,CXCL-6 may be down regulated. Such down regulation may occur when thecells are in normoxic conditions.

In some embodiments, under normoxic conditions one or more of IL-6,IL-8, TNF-α, IL-1, and VEGFA may be upregulated and one or more of NFκB,TGF-β, IL-10, CXCL6 and CXCL1 may be down regulated.

In some embodiments, under hypoxic conditions one or more of IL-6, IL-8,TNF-α, IL-1, NFκB, TGF-β, IL-10, VEGFA, CXCL6 and CXCL1 may beupregulated.

Upregulation or over-expression of a gene/protein comprises expressionof the marker at a level that is greater than would normally be expectedfor a cell or tissue of a given type. As such, upregulation may bedetermined by comparing the level of expression between virus infectedand non-infected cells of the same type.

Levels of expression may be quantitated for absolute comparison, orrelative comparisons may be made. Expression may be determined bymeasuring gene expression, e.g. by measurement of mRNA levels, or bymeasuring protein expression.

In some embodiments upregulation may be considered to be present whenthe level of expression in the test sample is at least 1.1 times that inthe control sample. In some embodiments the level of expression may beselected from one of at least 1.2, at least 1.3, at least 1.4, at least1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4 at least2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least3.0, at least 3.5, at least 4.0, at least 5.0, at least 6.0, at least7.0, at least 8.0, at least 9.0, or at least 10.0 times that in thecontrol sample.

Down regulation may be determined in a corresponding manner, e.g. insome embodiments down regulation may be considered to be present whenthe level of expression in the test sample is less than 0.9 times thatin the control sample. In some embodiments the level of expression maybe selected from one of less than 0.8, less than 0.7, less than 0.6,less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than0.1 times that in the control sample.

Accordingly, the infected cells represent a distinct and identifiablepopulation of cells, which may be useful in methods of adoptiveimmunotherapy (e.g. as described in Darcy et al., Current Opinion inImmunology 2014, 27:46-52 and in Andreesen et al., Journal of LeukocyteBiology Volume 64, October 1998, p419-426) in which the cell provides atherapeutic effect through its expression and/or secretion of definedfactors or cytokines which promote an immune response in the subject'sbody, preferably an anti-tumor response. This action of the cellpopulation is additional to its ability to transport oncolytic HerpesSimplex Virus to diseased tissue and release it in the tissue leading toa separate virus-mediated anti-tumor response.

Accordingly, in one aspect of the present invention a method isprovided, the method comprising obtaining a blood or tissue sample froma subject, separating monocyte, monocyte derived cells or macrophagesfrom said blood or tissue sample, infecting said cells with an oncolyticHerpes Simplex Virus, formulating a preparation comprising said infectedcells and administering said preparation to said subject. The method maybe part of a method of adoptive immunotherapy.

Magnetic Material

A magnetic material can include a material that is magneticallysusceptible, a magnetisable material or a material that can bemanipulated (e.g. moved) and/or positioned by a magnetic field. Themagnetic material can be non-magnetic but susceptible to manipulation orpositioning by a magnetic field, or be magnetic (e.g. a source of amagnetic field lines). As such, the magnetic material may be inherentlymagnetic or one which reacts, e.g. moves, in a magnetic field.

In preferred embodiments the magnetic material is a magneticallysusceptible particle or is a fluid, e.g. a fluid in which magneticallysusceptible particles are in suspension, often called a ferrofluid,

Magnetically susceptible particles can include magnetically susceptibleparticles, magnetisable particles or particles that can be manipulated(e.g. moved) and/or positioned by a magnetic field. The magneticallysusceptible particles can be non-magnetic but susceptible tomanipulation or positioning by a magnetic field, or be magnetic (e.g. asource of a magnetic field lines).

Typically the particles are of a size suitable to deliver the reagentinto the cell without causing damage to the cell. In one aspect, theparticles have a mean size of between 10 μm and 5 nm, such as between 1μm and 10 nm, for example between 200 nm and 20 nm or between 5 nm and50 nm. In another aspect the magnetically susceptible particles may bespherical beads and may have a diameter of at least about 0.05 μm, atleast about 1 pm, at least about 2.5 μm, and typically less than about20 μm, or may have a diameter of about 5 to 50 nm, 10 to 40 nm, 20 to 30nm or about 25 nm.

Not wishing to be limited by theory, it is believed that largerparticles will give improved uptake into monocytes, monocyte derivedcells or macrophages. For example, magnetite particles >30 nm willexperience a torque in an oscillating magnetic field as dictated by theformula r=μB sinθ, where r is the torque, μ is the magnetic moment, B isthe magnetic flux density and θ is the angle between the applied fieldand the particle's magnetization vector. For example, the precise amountof torque is influenced by the particle shape. The movement of theparticle induced by this torque is believed to ‘drag’ the particle intoand across the surface of the cell, inducing uptake of the particle byan endocytic mechanism. The uptake of the particle by normal cellularprocesses means that there is no mechanical damage to the cell (ascompared to, for example, biolistic methods or electroporation), thusimproving the rate of cellular survival post particle delivery.

A magnetically susceptible particle can be, for example, a magneticallysusceptible particle described, in U.S. Patent Application PublicationNos. 20050147963 or 20050100930, or U.S. Pat. No. 5,348,876, each ofwhich is incorporated by reference in its entirety, or commerciallyavailable beads, for example, those produced by Dynal AS (InvitrogenCorporation, Carlsbad, Calif. USA) under the trade name DYNABEADS™and/or MYONE™. In particular, antibodies linked to magneticallysusceptible particles are described in, for example, United StatesPatent Application Nos. 20050149169, 20050148096, 20050142549,20050074748, 20050148096, 20050106652, and 20050100930, and U.S. Pat.No. 5,348,876, each of which is incorporated by reference in itsentirety.

In one aspect the particle comprises a paramagnetic, superparamagnetic,ferromagnetic and/or antiferromagnetic material, such as elemental iron,chromium, manganese, cobalt, nickel, or a compound and/or a combinationthereof (e.g. manganese and cobalt ferrites). A particle may be asuper-paramagnetic iron oxide (SPIO) particle. For example, suitablecompounds include iron salts such as iron oxide, magnetite (Fe₃O₄),maghemite (γFe₂O₃), greigite (Fe₃S₄) and chromium dioxide (CrO₂).

The particles may comprise the magnetic material embedded in a polymer,for example within the pores of a polymer matrix. Alternatively, theparticles may comprise a magnetic core surrounded by a biocompatiblecoating, for example silica or a polymer such as dextran, polyvinylalcohol or polyethylenimine.

The magnetically susceptible particle may comprise a reagent. Thereagent may be associated with (e.g. conjugated to) the particle bycovalent or non-covalent bonds (for example, hydrogen bonding,electrostatic interactions, ionic bonding, lipophillic interactions orvan der Waals forces). In one aspect the reagent and particle arecovalently linked, for example by exposing the reagent to particlesbearing reactive side chains, for example benzidine for linking to thetyrosine residues of proteinaceous reagent, or periodate for linking tocarbohydrate groups. In another aspect the particle may be linked to amolecule with binding activity (e.g. avidin) and the reagent may belinked to a ligand of said binding molecule (e.g. biotin). This enablesthe particle and reagent to be easily conjugated in vitro. In a furtheraspect the particle may comprise the reagent absorbed into a matrix,such as a polymer matrix.

The magnetic material is preferably exogenous to the monocyte, monocytederived cell or macrophage, i.e. originating outside of the monocyte,monocyte derived cell or macrophage and optionally not being a materialthat is normally present in a monocyte, monocyte derived cell ormacrophage.

Application of a Magnetic Field

In embodiments where monocytes, monocyte derived cells or macrophagesare loaded with an exogenous magnetic material, following administrationof the monocytes, monocyte derived cells or macrophages to a subject amagnetic field may be applied to the subject in order to direct theadministered cells to a desired location in the subject's body, e.g. toa tumor. The cells are thereby subjected to a magnetic force, being theforce that is exerted on the magnetic material when it is in a magneticfield having a gradient. The magnetic force may cause the magneticmaterial to move toward the source of the magnetic field. The magneticforce may also cause the particle to experience a torque. In somearrangements, the magnetic force may cause the particle to move awayfrom the source of the magnetic field. This can occur if the particle ismagnetically blocked and unable to rotate.

As used herein, the ‘force field’ of a magnet, or of a magnet array,describes the volume of space surrounding the magnet or magnet array inwhich a magnetic material will experience a magnetic force.

The magnetic field may be provided by a magnet field source, typically amagnet, or array of magnets. The magnet may be an electromagnet. Thetype and size/power of magnet selected will depend on the application.For example, where cells are administered locally to tumor near thesurface of the body, e.g. a primary melanoma, a handheld magnet may besufficient to apply a suitable magnetic force to direct the cells, e.g.through tissue or blood vessels, toward the site of the tumor. In otherinstances, for example where the tumor is located deeper within the bodyand/or where administration is non-local to the tumor, e.g. systemicadministration, the subject may be placed in a variable or oscillating,and preferably controllable, magnetic field, such as that of anelectromagnet or that provided by a magnetic resonance imaging (MRI),nuclear magnetic resonance imaging (NMRI) or magnetic resonancetopography (MRT) apparatus.

In some preferred embodiments methods and uses according to the presentinvention involve the direction or targeting of monocytes, monocytederived cells or macrophages loaded with a magnetic material to aselected tissue or tumor that is not located near the skin, i.e. to adeep tissue or organ. A deep tissue or organ may be one that is at least4cm or 5cm or more away from the surface of the skin (measured asshortest distance to the surface of the skin from the centre of theregion of the tissue or tumor to be treated). The tissue or tumor may bein the core of the subject's body (i.e. the part of the body that doesnot include the legs and arms). The tissue or tumor may be in the head,neck, thorax, abdomen, or pelvis. The tissue or tumor may be one of themajor organs, or in one of the major organs, such as the adrenal gland,adrenal medulla, anus, appendix, bladder, bone, bone marrow, brain,breast, cecum, central nervous system (including or excluding the brain)cerebellum, cervix, colon, duodenum, endometrium, gallbladder,oesophagus, heart, ileum, intestines, jejunum, kidney(s), lacrimal glad,larynx, liver, lung(s), lymph, lymph node, mediastinum, mesentery,myometrium, nasopharynx, omentume, ovary, pancreas, parotid gland,peripheral nervous system, peritoneum, pleura, prostate, rectum,salivary gland, sigmoid colon, small intestine, spleen, stomach, testis,thymus, thyroid gland, or uterus. The monocytes, monocyte derived cellsor macrophages may be directed or targeted to a region of a tissue ortumor that is hypoxic.

To effect treatment, the subject may be administered cells loaded withthe magnetic material, and be positioned within a magnetic field. Themagnetic field may then be varied or otherwise manipulated relative tothe subject and/or to the cells so as to apply a magnetic force to themagnetic material contained within the cells, directing the magneticmaterial (and cells) toward a desired location in the subject's body,taking account as necessary of the architecture of the tissue, e.g.blood vasculature, to which the cells were administered.

Apparatus and techniques for magnetically guiding and/or localisingcells loaded with a magnetic material, and other agents, to a targetsite within the body (sometimes called Magnetofection) are known tothose of ordinary skill in the art, and are described, by way ofexample, in Muthana et al., (A novel magnetic approach to enhance theefficacy of cell-based gene therapies. Gene Therapy (2008) 15, 902-910);Polyak and Friedman, (Magnetic targeting for site-specific drugdelivery: applications and clinical potential. Expert Opinion on DrugDelivery, January 2009, Vol. 6, No. 1: Pages 53-70); Plank et al.,(Magnetically enhanced nucleic acid delivery. Ten years ofmagnetofection—Progress and prospects. Advanced Drug Delivery Reviews.Vol. 63, Issues 14-15, November 2011, pages 1300-1331); and in Li etal., (Targeting Cancer Gene Therapy with Magnetic NanoparticlesOncotarget. April 2012; 3(4):365-370).

In preferred embodiments, the application of a magnetic field to thebody of a subject is non-invasive and non-surgical. The magnetic fieldsource is normally external to the subject's body and in preferredembodiments does not physically contact the subject's body.

Cancer

A cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentume, oral cavity, ovary, pancreas, parotid gland, peripheralnervous system, peritoneum, pleura, prostate, salivary gland, sigmoidcolon, skin, small intestine, soft tissues, spleen, stomach, testis,thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, whiteblood cells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer, colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancerand sarcoma.

In some embodiments the cancer may be a solid tumor.

Hypoxia

In some embodiments, a tissue or cancer may be one that has a hypoxicenvironment and treatment may be directed to cells within thatenvironment, either together with surrounding normoxic cells orindependent of them.

Physiological normoxia varies between tissues, between animals andbetween individuals. A tissue or organ may have variable measurements,for example depending on the pattern of vascularisation. In general,healthy mammalian internal tissues and organs will typically have a meanaverage partial pressure of oxygen greater than 20 mmHg [about 2.62%oxygen] (e.g. about >35 mmHg in brain, >50 mmHg in intestinaltissue, >35 mmHg in liver, >25 mmHg in muscle)

Techniques for measurement of physiological normoxia and hypoxia in livesubjects are described in Carreau et al., (J. Cell. Mol. Med. Vol.15,No.6, 2011 pp.1239-1253), incorporated herein by reference. Theseinclude non-invasive and invasive techniques. Non-invasive techniquesinclude imaging techniques such as positron emission tomography (PET),magnetic resonance spectroscopy (MRS, e.g. ¹⁹F-MRI, blood oxygendependent-MRI, or dynamic contrast-enhanced MRI), near-infraredspectroscopy (NIRS), and electron paramagnetic resonance spectroscopy(EPR), optionally in conjunction with a hypoxia marker such as anitroimidazole (e.g. azomycin). Other techniques include use of apolarographic sensor (often considered the gold standard for measuringoxygen tension), an optical fibre-based sensor in conjunction with a pO₂sensitive fluorescent dye such as ruthenium chloride, and massspectrometry.

In some embodiments a method of treatment may comprise measuring ordetermining the state of normoxia and/or hypoxia in a tissue in asubject, e.g. measurement of oxygen partial pressure, and selecting thesubject and/or a tissue or part of a tissue in the subject, fortreatment with a monocyte, monocyte derived cell or macrophage infectedwith an oncolytic herpes simplex virus. In embodiments where the cellscontain an exogenous magnetic material and the determination of thestate of normoxia/hypoxia is made using a magnetic resonance method bothdetermination of the state of normoxia/hypoxia and direction of cellstowards the selected tissue or part of tissue may optionally beperformed simultaneously.

Hypoxia is known to occur in tumors. Rapid growth of the tumor withoutcorresponding angiogenesis or neovascularisation leads to regions of thetumor in which the oxygen concentration is lower than in normal(normoxic) healthy tissues. As such, hypoxic microenvironments developand the metabolism of the tumor cells may become adapted to the hypoxicenvironment. Tumor hypoxia is reviewed in Kizaka-Kondoh et al., (Tumorhypoxia: A target for selective cancer therapy. Cancer Sci December2003, vol. 94, no.12, 1021-1028) and Höckel and Vaupel (Journal of theNational Cancer Institute Vol. 93, No.4, Feb. 21, 2001, p266-276), eachof which are specifically incorporated by reference herein.

Tumor hypoxia is common in solid tumors. Many solid tumors contain areasof low O₂ partial pressure that cannot be predicted by clinical size,stage, grade or histology. In some embodiments hypoxia may be defined bya partial pressure of oxygen of one of less than about 20 mmHg, lessthan about 15 mmHg, less than about 14 mmHg, less than about 13 mmHg,less than about 12 mmHg, less than about 11 mmHg, less than about 10mmHg, less than about 9 mmHg, less than about 8 mmHg, less than about 7mmHg, less than about 6 mmHg, less than about 5 mmHg, less than about 4mmHg, less than about 3 mmHg, less than about 2 mmHg, or less than about1 mmHg. In some embodiments hypoxia may be defined as tissue having apartial pressure of oxygen in the range 0.01 to 15 mmHg, 5 to 15 mmHg,0.01 to 10 mmHg, 3 to 10 mmHg, 5 to 10 mmHg, 7 to 10 mmHg, 8 to 10 mmHg,8 to 11 mmHg, or 7 to 12 mmHg. In some embodiments hypoxia may bedefined as tissue having a partial pressure of oxygen of one of about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mmHg. [Note: 1 mmHg=133.322 Pa; 1%oxygen=1.013 kPa or about 7.64 mmHg]

As cellular metabolism may be adapted in hypoxic conditions, hypoxia mayalso be determined by measuring expression of one or more markers.

Such markers include transcription factors, e.g. NFκB or one of thehypoxia inducible factor (HIF) family, e.g. the isoforms HIF-1a, HIF-2α.HIF-1 has two subunits, HIF-1α and HIF-1β. HIF-1α and HIF-2α are inducedwithin minutes of occurrence of hypoxia. HIF-1α is primarily an acuteresponse to hypoxia and HIF-1α levels tend to reduce in prolongedhypoxia. HIF-2α levels tend to continue to increase with time inhypoxia. Induction of HIF-1α normally requires a lower pO₂(<5% [Carreauet al]) than is required for induction of HIF-2α. As such, in someembodiments hypoxia may be determined by measuring upregulation ofexpression of NFκB, HIF-1α or HIF-2α, which measurement may be comparedto a corresponding tissue considered to be at physiological normoxia.

Another group of markers are the hypoxia regulated microRNAs (HRMs),which include miR-21, 23a, 23b, 24, 26a, 26b, 27a, 30b, 93, 103, 106a,107, 125b, 181a, 181b, 192, 195, 210 and 213, which may be upregulatedin hypoxic cells. Some microRNAs may be down-regulated in hypoxic cells,such as miR-15b, 16, 19a, 20a, 20b, 29b, 30b, 30e-5p, 101, 141, 122a,186, 197, 320. As such, in some embodiments hypoxia may be determined bymeasuring upregulation, or downregulation, of one or more hypoxiaregulated microRNAs. Measurement may be compared to a tissue consideredto be at physiological normoxia.

Hypoxia may induce proteome changes, which in the case of tumor hypoxiamay promote tumor propagation and adaptation to the hypoxic environment.Such adaptation may involve adapting to nutritional deprivation, e.g. bystimulating transcription of glycolytic enzymes, glucose transporters(e.g. GLUT1 and GLUT3), angiogenic molecules, survival and growthfactors (e.g. VEGF), angiogenin, PDGF-β, or TGF-β.

Subjects

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient. A subject may have beendiagnosed with a cancer, or be suspected of having a cancer.

Kits

In some aspects of the present invention a kit of parts is provided. Insome embodiments the kit may have at least one container having apredetermined quantity of oncolytic Herpes Simplex Virus, e.g.predetermined viral dose or number/quantity/concentration of viralparticles. The oncolytic Herpes Simplex Virus may be formulated so as tobe suitable for infection of cells. The kit may further comprise atleast one container having a predetermined quantity of magneticmaterial.

The kit may be provided together with instructions for the infection ofmonocytes, monocyte derived cells or macrophages with the oncolyticHerpes Simplex Virus and/or for the loading of monocytes, monocytederived cells or macrophages with the magnetic material. Suchinstructions may be for carrying out said infection and/or loading exvivo or in vitro, e.g. under conditions of in vitro cell culture.

Methods according to the present invention may be performed, or productsmay be present, in vitro, ex vivo, or in vivo. The term “in vitro” isintended to encompass experiments with materials, biological substances,cells and/or tissues in laboratory conditions or in culture whereas theterm “in vivo” is intended to encompass experiments and procedures withintact multi-cellular organisms. “Ex vivo” refers to something presentor taking place outside an organism, e.g. outside the human or animalbody, which may be on tissue (e.g. whole organs) or cells taken from theorganism.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1. Chart showing titration of human macrophages at various timesafter infection with 4 pfu/cell HSV1716 and culture in normoxia orhypoxia. Approximately 300,000 primary human macrophages were infectedwith 1,180,000 pfu HSV1716 with samples collected at various times postinfection and HSV1716 titrated on Vero cells. Total titratable virus wasgraphed against time and the dotted line represents the amount of inputvirus

FIG. 2. Chart showing output (total pfu) from human macrophages after 72hrs of normoxia infection with HSV1716 at various input moi.Approximately 300,000 primary human macrophages were infected HSV1716 atmoi 40, 4, 0.4 and 0.04 with samples collected at 72 hrs post infectiononly and HSV1716 titrated on Vero cells.

FIGS. 3A-B. Representative density plots of LNCaP cell population after72 hours from infection at MOI 0 (control) (A) and 5 (B). BL3-A detector(X-axis) is a measure of PI, while BL1-A detector (Y-axis) is a measureof GFP. Each dot represents a cell. Quadrant R4 shows alive populationnot infected by HSV1716 (PI−/GFP−); quadrants R3 and R5 represent thequantity of dead cells (PI+); R2 shows living cells infected by HSV1716(PI−/GFP+). While in the control (A) the cell population is mainlydistributed in R4, at MOI 5 (B) cells move towards the right-hand side(R3+R5) and the upper side of the plot (R2), indicating respectivelyincreased cell death and presence of a percentage of living cellsinfected by HSV1716. Plots have been obtained from the Attune cytometricsoftware.

FIGS. 4A-B. HSV1716 induces LNCaP cell oncolysis. (A) X-axis shows MOI,Y-axis shows the percentage of living cells that have taken up HSV1716.Statistical significance was observed in normoxic conditions (left handbar of each data point; hypoxic=right hand bar of each data point) atboth MOI 0.5 and 5. (B) X-axis displays MOI, while percentage of celldeath is reported on Y-axis. Cell death is statistically significant atMOI 5 in both normoxic and hypoxic conditions (normoxic=left hand bar ofeach data point; hypoxic=right hand bar of each data point). Of note,data are the mean±SEM of n=4 repeats. p-value<0.05, measured by usingtwo-way Anova test for multiple comparisons.

FIGS. 5A-B. Representative density plots of PC3 cell population after 72hours from infection at MOI 0 (control) (A) and 5 (B). BL3-A detector(X-axis) is a measure of PI, BL1-A detector (Y-axis) is a measure ofGFP. Each dot represents a cell. Quadrant R4 shows alive population notinfected by HSV1716 (PI−/GFP−); quadrants R3 and R5 represent thequantity of dead cells (PI+); R2 shows living cells infected by HSV1716(PI−/GFP+). Compared to the control (A), where cell population is mostlycomposed by living cells (events distributed in R4), at MOI 5 (B) aconsistent proportion of cells has shifted towards the right-hand side(dead cells) and the upper side of the plot (R2: infected living cells).Plots have been obtained from the Attune cytometric software.

FIGS. 6A-B. HSV1716 has cytotoxic effect on PC3. (A) X-axis shows MOI,Y-axis shows the percentage of living cells that have taken up HSV1716.Statistical significance was observed in normoxic conditions (left handbar of each data point; hypoxia=right hand bar of each data point) atboth MOI 0.5 and 5. (B) X-axis displays MOI, Y-axis shows percentage ofcell death. Cell death is statistically significant at MOI 0.5 and 5 inboth normoxic and hypoxic conditions (normoxic=left hand bar of eachdata point; hypoxic=right hand bar of each data point). Results are themean±SEM of 4 (A) and 8 (B) repeats. p-value <0.05, measured by usingtwo-way Anova test for multiple comparisons.

FIGS. 7A-B. Representative density plots of T47D cell population after120 hours from infection at MOI 0 (control) (A) and 5 (B). BL3-Adetector (X-axis) is a measure of PI, BL1-A detector (Y-axis) is ameasure of GFP. Each dot represents a cell. Quadrant R4 shows alivepopulation not infected by HSV1716 (PI−/GFP−); quadrants R3 and R5represent the quantity of dead cells (PI+); R2 shows living cellsinfected by HSV1716 (P1−/GFP+). Living cell population (A, control)moves towards the right-hand side of the plot after 120 hours frominfection (B, MOI 5), indicating the presence of dead cells. A markedproportion of cells was also observed in the R2 quadrant (infectedliving cells). Plots have been obtained from the Attune cytometricsoftware.

FIGS. 8A-B. HSV1716 infection induces T47D cell death. (A) X-axis showsMOI, Y-axis shows the percentage of living cells that have taken upHSV1716. Statistical significance was observed in normoxic and hypoxicconditions (normoxic=left hand bar of each data point; hypoxia=righthand bar of each data point) at both MOI 0.5 and 5. (B) X-axis showsMOI, Y-axis shows percentage of dead cells. Cell death is statisticallysignificant at MOI 5 in both normoxic and hypoxic conditions. Resultsare the mean±SEM of n=3 (A) and n=5 (B) independent experiments. p-value<0.05, measured by using two-way Anova test for multiple comparisons.

FIGS. 9A-C. Representative density plots of MDM cell population after 96hours from infection at MOI 0 (control) (A), 5 (B) and 50 (C). BL3-Adetector (X-axis) is a measure of PI, BL1-A detector (Y-axis) is ameasure of GFP. Each dot represents a cell. Quadrant R4 shows alivepopulation not infected by HSV1716 (PI−/GFP−); quadrants R3 and R5represent the quantity of dead cells (PI+). Compared to the control (A),where cell population is mostly composed by living cells, at MOI 5 (B) aswitch towards the right-hand side of the graph is observed (R3, R5),indicating an increase in the percentage of dead cells. At MOI 50 (C), aslight increment in cell death is detected, however there is not aconsiderable difference between MOI 5 and 50. Plots have been obtainedfrom the Attune cytometric software.

FIG. 10. HSV1716 causes MDM cell death. X-axis shows MOI, Y-axis showspercentage of dead cells. Cell death is statistically significant at MOI5 and MOI 50 (p-value<0.001) in both normoxic and hypoxic conditions(normoxic=left hand bar of each data point; hypoxia=right hand bar ofeach data point). At MOI 50, an increment in cell death is observedunder hypoxic conditions. Results are the mean±SEM of n=4 independentexperiments. Statistical comparisons performed using two-way Anova testfor multiple comparisons.

FIG. 11. Table 1: Concentration of HSV1716 (PFU/ml) detected inmacrophage-conditioned medium. The table display the concentration ofHSV1716 (PFU/ml) present in supernatant collected from MDM infected atMOI 0, 5, 50 and incubated under normoxic and hypoxic conditions. Rowsshow the different microenvironment (normoxia, hypoxia); columnsindicate the viral infection performed (control, MOI 5, MOI 50). Viralparticles were detected at both MOI 5 and 50, with greater concentrationat MOI 50 in both normoxic and hypoxic conditions. Data are the mean±SEMof n=4 independent experiments.

FIG. 12. HSV1716 lyses human macrophages and is released into themicroenvironment. X-axis shows MOI at which MDM were infected, Y-axisshows concentration (PFU/ml) of HSV1716 found in supernatant collectedfrom plates. Results are statistically significant at MOI 50 underhypoxic conditions (p-value<0.0001, determined by using the two-wayAnova test for multiple comparisons). Data show the mean±SEM of n=4independent experiments. Normoxic=left hand bar of each data point;hypoxic=right hand bar of each data point.

FIG. 13. Viral particles contained in MDM-conditioned medium induceLNCaP cell death. X-axis shows the MOI used to infect macrophages fromwhich the conditioned medium was collected. Y-axis shows percentage ofdead cells. Cell death is statistically significant at MOI 50 innormoxic conditions (p-value<0.01). Results are the mean±SEM of n=3independent experiments. Statistical analysis was performed by usingtwo-way Anova test for multiple comparisons. Normoxic=left hand bar ofeach data point; hypoxic=right hand bar of each data point.

FIG. 14. Table 2: HSV1716 induces alterations in MDM gene expressionlevels. Table 2 shows fold changes in MDM gene expression calculated for10 genes of interest (named in the first row) after 48 hours fromHSV1716 infection at MOI 50 in both normoxic (second row) and hypoxic(third row) conditions. Infection under each condition has been repeatedtwice. Underlined values represent indicatively relevant alterations ingene expression, either up-regulation (value>1) or down-regulation(value<1); i.e. the underlined value 5.13 means that, after infection atMOI 50, the cytokine IL-8 was found to be overexpressed in hypoxicconditions, resulting in a 5-fold increase compared to the control.

FIG. 15. Infiltration of spheroids with infected MDM (MOI 50) inducesspheroid cell death. Graph shows cell death detected by flow cytometryafter 5 days from infiltration (day 9 of experiment). Y-axis shows %cell death (PI+), X-axis shows the MOI at which MDM were infected beforeinfiltration. While no significant differences in viability wereobserved between control spheroids (control) and infiltration withnon-infected MDM (0), cell death was statistically significant whenspheroids were infiltrated with MDM infected at MOI 50 (50) (51±5.92%cell death) when comparing the latter with both control and 0(p-values=0.009, 0.004 respectively). Results are the mean±SEM of n=3independent experiments. Statistical analysis was performed usingmultiple t tests.

FIG. 16. Possible use of MR targeting to steer cell-based therapies tospecific tissues in patients. (a) Schematic illustration: The cells usedfor these studies are derived from monocytes isolated from patientblood. These cells are cultured in the presence of various stimuli toproduce ‘therapeutic’ macrophages (e.g. cytokines, therapeutic genes orviruses) and loaded with superparamagnetic iron oxide particles (SPIOs)before re-infusion back into the same patient. (b) Schematicillustration: The subject is then placed in the centre of an MRI scannerwhere linear spatial encoding magnetic gradients can be used to induce aforce on a magnetized body. The magnetic cells injected into thebloodstream of the subject circulate and are targeted into the diseasedorgan/tissue under the influence of the applied magnetic field. Fieldmap plots demonstrate that significant field gradients can be generatedin various directions by the MRI gradient coils. The resulting magneticfield (dB/dy field) can steer magnetic cells towards the diseased tissuefor increased cell uptake.

FIGS. 17A-E. Magnetic macrophages were steered into primary prostatetumors using MR Targeting. (a) Schematic of targeted regions usingimaging gradients for MR Targeting. A -Y gradient is applied equallyacross the animal to target the location of the prostate as depicted(darkly shaded box). Three million magnetically labelled macrophageswere then administered to mice via i.v. injection and anesthetised micewere then placed into the isocentre of a 7 T MRI scanner. Subjects weresplit into 2 groups. Group 1 were imaged after 1 hour (no MR Targeting).Group 2 underwent MRI targeting. Post mortem the increased levels ofhuman macrophage uptake was confirmed by (b) FACS analysis ofcollagenase-treated tumors one hour after MRI targeting, and (c)histological staining of paraffin wax-embedded tumor sections with ananti-human CD68 antibody and Prussian blue (for SPIOs). RepresentativeRARE images and R2 maps for each group are shown (d) and (e). Bars=200μm.

FIGS. 18A-C. Magnetic macrophages were steered in to pulmonarymetastasis using MR Targeting. Short-pulsed magnetic gradients were usedto steer SPIO-loaded macrophages towards the lungs. (a) FACS analysis ofcollagenase-treated lungs showed significantly more human CD14+macrophages were present in lungs with rather than without MR targeting.(b) This was accompanied by increased immunostaining for human CD68 andPrussian blue in lung sections. (c) Immunostaining with CD31 and H&Eindicated that MR targeted delivery of magnetic macrophages into thelungs had no adverse affects on the lung vasculature compared todelivery without targeting. Representative data are shown from one oftwo replicate experiments in which n=3 mice/group. SEMs are depicted.*P<0.01 compared with non-MR targeted lungs in panel A. Bar=50 μm.

FIGS. 19A-F. Magnetic targeting increases the anti-tumor effects ofmacrophage virotherapy on human prostate (LUC-LNCaP) tumors.Tumor-bearing mice were administered with a single dose of humanmonocyte-derived macrophages (MDM) carrying the oncolytic virus, HSV1716(MDM+OV). These were divided into three groups of mice (each with 5mice/group). One group underwent MR targeting to either the prostategland or lungs (MDM+OV+MRT) for 1 h, another was exposed to the MRIscanner but with no MR targeting (MDM+OV no MRT) and the third (MDM+OV)did not enter the MRI scanner. Additional groups of mice received 100 ulof PBS (Control), a single dose of 1×10⁷ pfu HSV1716 (OV) or 3 millionuntreated MDM. Mice were imaged weekly using the IVIS imaging systemand, after 21 days, tumors and lungs were removed and processed forhistology. (a) Tumor luminosity showed MR Targeting significantlyimproved the effect of OV-MDM on tumor growth (b) Representative IVISimages and photographs of primary tumors following various treatments(c) Representative RARE images for MDM+OV with or without MR targetingshow marked difference in tumor size at the beginning and end of therapy(d). Appearance of H&E stained sections to show (e) the presence ofnecrosis in primary tumors and (f) metastases in the lungs of micereceiving MDM+OV with or without MR targeting. Corresponding data fromall groups are shown (e). Data shown are means+/−SEMs. Quantitativeanalysis was carried out on 10 high-power fields (HPF; x20magnification) per tissue section. Statistical significance differences,*P<0.05; **P<0.001; ***P<0.0001 compared with MDM+OV+MRT to MDM+OV (noMR targeting) and {circumflex over ( )} comparing MDM+OV (no MRtargeting) and free OV group; Bar, 200 μm.

FIGS. 20A-C. Initial MRT investigations using a novel trans-endothelialmigration (TEM) flow assay. A flow chamber that can accommodate 3D tumorspheroids as well as a vascular endothelial layer was designed. Theflowing ‘magnetic cells’ will therefore need to cross the vascularbarrier before entering a 3D tumor simulating the passage of cellsacross endothelial cells in a blood vessel wall (A:Left Panel). The TEMflow chamber is placed in the iso-centre of an MRI scanner with aspherical (6 mm diameter) homogenous 7 T magnetic field. A pulsedgradient (50% of max) with strength of 300 mT/m in the (-y) gradientdirection was applied. The resulting heterogeneous magnetic field (dB/dyfield) can steer magnetic particles towards the tumor spheroids forincreased uptake (A:Right panel). (B) Graph showing the effect of theSPIOs on cell death; Uptake was confirmed by a distortion in the MRIimage and a loss of signal compared to when no MRT was applied (Ci).Corresponding fluorescent images of whole spheroids infiltrated withmacrophages carrying a reporter adenovirus (AdCMVGFP) are shown in(Cii). Flow cytometry of enzymatically dispersed spheroids revealed thatthe number of magnetic-cells infiltrating spheroids (% of all cellspresent in spheroids that were CD14+) was significantly (*P<0.03)increased when a gradient was applied (Ciii &iv). Data are Means±SEM andare representative of 6 replicate experiments. Statistical significancedifferences p=0.0001, compared with MRT treated cells. Bar=100 um.

FIGS. 21A-D. Magnetic macrophages were steered into primary prostatetumors using magnetic fields generated in an MRI scanner. Three millionmagnetically labelled macrophages were administered via i.v. injectionand mice were then placed into the isocentre of a 7 T MRI scanner.Subjects were split into 2 groups. Group 1 were imaged after 1 hour (noMRT). Group 2 underwent MRT. The number of vessels per high power fieldper view was recorded in sections of CD31 labelled tumors (a). Steeringof macrophages into the tissue using MRT had no significant effect onvessel numbers in tumors (p=0.5165) compared to mice who received noMRT. Representative MRI images for each group following MRT into tumorsshow a qualitative decrease in signal and this was confirmed by analysisof the transverse relaxation rate in both groups (b). Group 2 shows anincreased decay rate over group 1. The estimated best echo time forlooking at signal differences with MRI is around 60 ms—MRI steeringleads to a 10% decrease in signal at this echo time. This significantsignal decrease suggests the presence of increased levels of ironin-group 2. The normal decay rate of tumor tissue is also shown forcomparison (Control). This experiment was repeated but using macrophageswithout SPIOs (N=3 mice per group). Very little distortion was visiblein the MRI images of tumors in (c), indicating low uptake of nonmagneticmacrophages and this was confirmed by FACS analysis of collagenasetreated tissue (d). Data are presented are the means±SEM. Bar=200 um.

FIG. 22. Magnetic macrophages were detected in very low numbers in othertissues/organs. MRI steering of magnetic macrophages into tumorsresulted in very few macrophages localising to other tissues. This wasdetermined by histological staining of paraffin-wax embedded sections oftissues and organs removed post-mortem. Representative sections of theliver and spleen taken from tumor-bearing mice that received MRT or noMRT are shown. In both these tissues few human macrophages (<2%, Liver &<1% Spleen) following staining with anti-CD68 were detected. Bar=100 um

FIGS. 23A-B. Magnetic macrophages were steered into areas of pulmonarymetastasis using MRT. This was confirmed by histological staining ofwax-embedded sequential sections of lung tissue with EPCAM (to detectthe human prostate tumor cells) and Prussian Blue (PB) to detect theiron-positive human macrophages. Representative images show thatmacrophages positive for PB were detected in close proximity to themetastatic deposits within the lungs of mice following MRI steering (a).Of note, the iron with macrophages targeted to the lungs by MRT was alsovisible following H&E staining (b). Bar=200 um and Bar=50 um.

FIGS. 24A-D. Graphs showing HSV1716 induces LNCaP and macrophageoncolysis. HSV1716:GFP was added to cultures of LNCaP cells incubated innormoxic (20% O₂) and hypoxic (0.5% 02) culture conditions. Tumour celldeath was assessed by flow cytometry using propidium iodide and wassignificantly increased over uninfected cells (a). This was dosedependent and in normoxia at MOI 5 p<0.03 at MOI 50 p<0.001 & in hypoxiaat MOI 5 p<0.01 at MOI 50 p<0.001. No statistical significance wasobserved between normoxic and hypoxic conditions at both MOI 5 and 50.HSV1716 is effectively taken up by MDM at MOI 5 and 50 as assessed byflow cytometry 48 h post infection. Normoxic culture conditions resultedin significantly more GFP expressing macrophages at MOI 5 (p<0.0004) andMOI 50 (p <0.001) compared to hypoxic conditions (b) but interestinglythe concentration of HSV1716 (PFU/ml) detected in macrophagesupernatants 96 h following infection was greater at both M015 and 50 inhypoxia compared to normoxic conditions. Finally, macrophage cell deathwas equally infective in both normoxia and hypoxia (p<0.2) followinginfection with HSV1716 (c, d). Data are the mean±SEM of n=4 independentexperiments.

FIGS. 25A-B. HSV1716 infects, replicates in and kills human macrophages.Day 7 human monocyte-derived macrophages infected with GFP taggedHSV1716 demonstrate a significant increase in infection which correlateswith an increase in cell death. Charts show (A) infection of humanmonocyte-derived macrophages, (B) macrophage death. All data werenormalised to the house keeping gene GAPDH and 6 independent experimentswere performed (n=6). X-axis 0=macrophages (no virus).

FIGS. 26A-C. HSV1716 replication within human macrophages. Investigationof the expression of viral proteins showed that both immediate early(ICP0) and late (gB) genes required for viral replication demonstratesignificant gene expression in macrophages. Charts show (A) ICP0expression, (B) ICP8 expression, (C) gB expression. All data werenormalised to the house keeping gene GAPDH and 6 independent experimentswere performed (n=6). X-axis 0=macrophages (no virus).

FIGS. 27A-D. Mechanism of cell death in human macrophages. HSV1716 killsmacrophages via apoptosis and in a Fas dependent manner with both FasLand BcI-2 gene expression up-regulated 24 hours after infection withHSV1716 at an MOI of 5. Expression of genes involved in autophagy (Atg5and LC3B) were not significantly altered. Charts show expression of (A)FasL, (B) BcI-2, (C) LC3B, (D) Atg5. All data were normalised to thehouse keeping gene GAPDH and 6 independent experiments were performed(n=6). X-axis 0=macrophages (no virus).

FIGS. 28A-F. HSV1716 infection induces an inflammatory phenotype inmacrophages. HSV1716 infection of day 7 monocyte-derived macrophagessignificantly induces mRNA expression of typical markers of inflammation24 hours post infection. Charts show expression of mRNA for (A) IL-6,(B) IL-8, (C) IL-10, (D) TNFalpha, (E) TGFbeta, (F) NFkappaB. All datawere normalised to the house keeping gene GAPDH and 6 independentexperiments were performed (n=6). X-axis 0=macrophages (no virus).

FIGS. 29A-E. HSV1716 infection induces an inflammatory phenotype inmacrophages. HSV1716 infection of day 7 monocyte-derived macrophagessignificantly induces mRNA expression of typical inflammatory M1macrophage markers (NOS2, CXCL10) and down regulates typical M2 markersexpressed by tumour-derived macrophages (MRC1). Charts show mRNAexpression of (A) Arg1, (B) Nos2, (C) MRC1, (D) VEGF, (E) CXCL10. Alldata were normalised to the house keeping gene GAPDH and 6 independentexperiments were performed (n=6).

FIG. 30. Chart showing HSV1716 infection induces PCNA expression inmacrophages. HSV1716 infection of day 7 monocyte-derived macrophagessignificantly induces PCNA expression. This is a potential mechanism forinducing viral replication and macrophage cell death in non-tumour cellsthat are terminally differentiated. All data were normalised to thehouse keeping gene GAPDH and 4 independent experiments were performed(n=4).

The details of one or more embodiments of the invention are set forth inthe accompanying description below including specific details of thebest mode contemplated by the inventors for carrying out the invention,by way of example. It will be apparent to one skilled in the art thatthe present invention may be practiced without limitation to thesespecific details.

EXAMPLES

The examples presented below show that tumour-conditioned macrophagesinfected with oncolytic HSV1716 (Seprehvir) display a classic activated(M1) profile characterized by the expression of pro-inflammatory factorssuch as iNOS, IL-6, IL-8 and TNF-α. Furthermore, the M1 macrophages canbe magnetically labeled using super-paramagnetic iron oxidenanoparticles (SPIOs) and then non-invasively steered from thebloodstream into deep target tissues, including primary and secondarytumours, using pulsed magnetic-field gradients inherent to all magneticresonance imaging systems (MRI). We have used this magnetic resonancetargeting (MRT) approach to deliver a cell-based oncolytic virotherapy.Relaxometry measurements suggest that standard MR imaging can then beused to monitor the efficacy of this therapy.

Example 1 HSV1716 and Human Primary Macrophages

1) In an initial study human macrophages were infected with HSV1716 atapproximately 4 pfu/cell and the cells were then incubated under normaland hypoxic conditions. Samples were removed at various time pointsafter infection (+1.5 hr, +24 hrs, +48 hrs and +72 hrs) and titrated(FIG. 1).

Within 1 hour 90% of the virus had been adsorbed by the macrophages andthen no virus was detectable at 24 or 48 hrs in either normoxia orhypoxia (detection limit of titration is 100pfu/ml).

Significantly, virus was detectable at 72 hrs but the amounts at thistime were similar in the normoxic vs hypoxic macrophages. This emergentvirus is of significant interest as it could either be the originalinput which had entered some transient latent state or represent thefirst wave of replication in the macrophages.

2) Macrophages were infected with decreasing HSV1716 moi (40, 4, 0.4 and0.04) and samples were titrated after 72 hrs only. Virus was detectedfrom the macrophages infected at moi 40, 4 and 0.4 but not from thoseinfected at 0.04 moi (FIG. 2). Interestingly, the ratio of virusdetected after 72 hrs relative to the input pfu was approximately thesame and similar to those from the two other 72 hr normoxia/hypoxia timepoints shown in FIG. 1.

In summary, human primary macrophages were found to have a high capacityto adsorb HSV1716, and active virus can be recovered from themacrophages after 48 hrs in culture.

Example 2

Three different cancer cell lines were used (LNCaP, PC3, T47D) andmacrophages were derived from human mononuclear cells; multicellularspheroids were prepared on agarose-coated culture plates; HSV1716GFP wasused, to allow quantification of uptake by cancer cells and tumorspheroids by fluorescence microscopy and flow cytometry; finally, RT-PCRwas performed to analyse changes in macrophage gene expression afterHSV1716 infection.

Results showed that HSV1716 induces cell death in prostate and breastcancer cell lines and that macrophages infected by HSV1716 areeffectively killed within 96 hours; moreover, infiltration of spheroidswith HSV1716-infected macrophages causes tumor spheroid cell death.

Materials and Methods Cell Lines

Human prostate carcinoma cell lines LNCaP and PC3 and human breastcarcinoma cell line T47D were provided by Dr Helen Bryant (Department ofOncology, The Medical School, Sheffield, UK). Cells were cultured inRPMI supplemented with 10% Fetal Bovine Serum.

Preparation of Human MDM

Macrophages were derived from human mononuclear cells, which wereisolated from platelet-depleted buffy coats (Blood Transfusion Service,Royal Hallamshire Hospital, Sheffield, UK). Mononuclear cells wereseparated from blood by using Ficoll gradient centrifugation (BURKE2003). After isolation, mononuclear cells were seeded into T75 tissueculture flasks (˜70×10⁶ cells/flask) and cultured in IMDM supplementedwith 2% AB serum for 3 days.

Herpes Simplex Virus 1716

HSV1716 was provided by Virttu Biologics (Glasgow, UK). Infection oftumor cells was performed by using multiplicity of infection (MOI), thatis, the number of virus particles added per cell during infection, of0.5 and 5. For macrophage infection, MOI of 5 and 50 were used. Thelabelling with GFP allowed detection of HSV1716 (measured by flowcytometry and fluorescent microscopy).

Infection of Primary MDM

MDM were cultured for 3 days in IMDM supplemented with 2% AB serum;after 3 days, cells were washed with PBS and medium was replaced withRPMI supplemented with 10% FBS. Cells were infected at M01 50 andincubated overnight (normoxic conditions: 20% pO₂; hypoxic conditions:0.1% pO₂). After 24 hours, conditioned medium was replaced with freshmedium and cells were incubated for further 72 hours. After 96 hours (4days) from infection, cell viability was measured by flow cytometry.

Infiltration of Primary MDM into Tumor Spheroids

Tumor spheroids were prepared using LNCaP cells by seeding 2×10⁴cells/well into 2% agarose-coated 96-well plates in 100 μl RPMI (+10%FBS). After 72 hours (3 days), 5×10³ infected macrophages were added toeach well. Analysis of cell death was performed after a further 5 days;each day, spheroids were observed under the fluorescence microscope todetect the presence of HSV1716GFP in the hypoxic core.

Flow Cytometry

Cell viability/death and GFP expression were measured by flow cytometry.Cells were harvested, re-suspended in PBS and labelled with PI (1pl/sample) to quantify cell death. Attune Acoustic Focusing Cytometer(Life Technologies) was used to analyse percentage of Pl positive cellsand GFP positive cells in each sample. PI (excitation wavelength: 488nm; maximum emission: 617 nm) was detected by BL3 detector; BL1 was usedfor GFP detection (excitation at 488 nm and maximum emission at 509 nm).

RT-PCR

Reverse transcription-polymerase chain reaction (RT-PCR) was performedto detect RNA levels in infected macrophages and determine whetherHSV1716 causes changes in gene expression. Cells (1.5×10⁶) were platedinto 6-well plates and infected with HSV1716 at MOI 50. After incubationfor 48 hours (normoxic conditions: 20% pO₂; hypoxic conditions: 0.1%pO₂), cells were harvested and RNA extraction was performed using theRNeasy Mini Kit (Qiagen). cDNA was synthesised from RNA using the Primerdesign Precision nanoScript RT Kit and the T100 Thermal Cycler(Bio-Rad). cDNA was plated in 384-well PCR plates with primers of genesof interest. PCR was performed using the ABI7900 Real Time PCR.

IL-6 forward: (SEQ ID NO: 1) 5′-CGAAAGTCAACTCCATCTGCC-3′ reverse:(SEQ ID NO: 2) 5′-GGCAACTGGCTGGAAGTCTCT-3′ IL-8 forward: (SEQ ID NO: 3)5′-GGGCCATCAGTTGCAAATC-3′ reverse: (SEQ ID NO: 4)5′-TTCCTTCCGGTGGTTTCTTC-3′ TNFα forward: (SEQ ID NO: 5)5′-CCAGGAGAAAGTCAGCCTCCT-3′ reverse: (SEQ ID NO: 6)5′-TCATACCAGGGCTTGAGCTCA-3′) IL-1 forward: (SEQ ID NO: 7)5′-CACCTCTCAAGCAGAGCACAG-3′ reverse: (SEQ ID NO: 8)5′-GGGTTCCATGGTGAAGTCAAC-3′) NFκB forward: (SEQ ID NO: 9)5′-ACCTGAGTCTTCTGGACCGCTG-3′ reverse: (SEQ ID NO: 10)5′-CCAGCCTTCTCCCAAGAGTCGT-3′ TGFβ forward: (SEQ ID NO: 11)5′-TAGGAACAGGCGGCGACGAATACA-3′ reverse: (SEQ ID NO: 12)5′-CACAATCACAAGGCAACTTCAAT-3′ IL-10 forward: (SEQ ID NO: 13)5′-GCCTAACATGCTTCGAGATC-3′ reverse: (SEQ ID NO: 14)5′-CTCATGGCTTTGTAGATGCC-3′ VEGF-A forward: (SEQ ID NO: 15)5′-GAAGTTCATGGACGTCTACCAG reverse: (SEQ ID NO: 16)5′-CATCTGCTATGCTGCAGGAAGCT-3′ CXCL-6 forward: (SEQ ID NO: 17)5′-GAATTTCCCCAGCATCCCAAAG-3′ reverse: (SEQ ID NO: 18)5′-TGCCTTCTGCACTCCCTTTATC-3′ CXCL-1 forward: (SEQ ID NO: 19)5′-AGAATGTTTTCAAATGTTCTCCAGTC-3′ reverse: (SEQ ID NO: 20)5′-GGCCATTTGCTTGGATCCG-3′

Statistical Analysis

Data are reported as mean±SEM. Statistical analysis and graphics wereperformed using GraphPad Prism. Two-way ANOVA test for multiplecomparisons and multiple t tests were performed to compare experimentaldata obtained. Statistical significance was limited to the value ofp=0.05.

Results HSV1716 Induces Tumor Cell Death LNCaP Cell Line

To analyse the oncolytic potentiality of HSV1716 on prostate cancercells, LNCaP cells were seeded into 12-well plates (2×10⁴ cells/well)and infected with HSV1716 at MOI 0 (control), 0.5 and 5. The infectionwas repeated using HSV1716-GFP in order to visualise virus uptake byliving cells. Plates were kept in normoxic and hypoxic incubators, toinvestigate the ability of HSV1716 to kill cells in both oxygenated andnon-oxygenated conditions. After 24 hours, conditioned medium wasreplaced with fresh medium. After 72 hours, plates were harvested andcells were analysed by flow cytometry. Cell death was measured as PI(+)cells; virus uptake in living cells was measured as GFP(+)/PI(−) cells(FIG. 3). After 72 hours from infection, virus uptake was observed in56±6.35% of cells at MOI 0.5 (p<0.0001) and 53±8.7% of cells at MOI 5(p<0.0001) in normoxic conditions, while levels were considerably lowerin hypoxic conditions (16±4.73% at MOI 5, p<0.05). At MOI 5,statistically significant levels of cell death were observed in bothnormoxic (31±7.32%, p<0.01) and hypoxic (38±1.36%, p<0.05) conditions(FIG. 4). Interestingly, although virus uptake did not seem to be thathigh under hypoxic conditions, results revealed significant levels ofcell death at MOI 5 (FIG. 4).

PC3 Cell Line

PC3s are a prostate cancer cell line with high metastatic capacity andconsiderably more aggressive than LNCaPs. 2×10⁴ cells were seeded into12-well plates and infected with HSV1716 at MOI 0, 0.5 and 5. Cells wereincubated in normoxic and hypoxic conditions for 72 hours. After 24hours, conditioned medium was replaced with fresh medium to eliminateviral particles not taken up by cells. After 72 hours, cells wereanalysed by flow cytometry and the amount of PI(−)/GFP(+) cells wasplotted (FIG. 5). Percentages of virus uptake indicate a significantpresence of living cells infected by viral particles in normoxicconditions (20±4.46% at MOI 0.5, 17±4.37% at MOI 5, p<0.05); cell deathwas statistically significant in both normoxic (23±2.49% at MOI 0.5,p<0.0001, and 17±3.14% at MOI 5, p<0.01) and hypoxic conditions(20±1.34% at M010.5 and 19±2.68% at MOI 5, p<0.05). Percentages of virusuptake by living cells and cell death were reported graphically (FIG.6).

T47D Cell Line

To investigate the oncolytic ability of HSV1716 on different types ofsolid tumors, effects of infection of a breast carcinoma cell line,T47D, was evaluated. 1×10⁵ cells were seeded into 12-well plates andinfected with HSV1716 and HSV1716-GFP at MOI 0, 0.5 and 5. Cells wereincubated under normoxic and hypoxic conditions for 72 hours andanalysed by flow cytometry. No signs of cell death were observed in anyconditions, while virus uptake was markedly high even at MOI 0.5 (datanot shown). Therefore, the analysis was repeated after 120 hours, toverify whether T47D cell line is either not responsive to the HSV1716,or just less sensitive than prostate carcinoma cell lines. After 120hours, cells were observed by flow cytometry and the amount ofPI(−)/GFP(+) cells was plotted (FIG. 7). Virus uptake by living cellswas significant at MOI 0.5 and 5, with considerably higher levels innormoxia (38±0.35% at MOI 0.5, 49±0.49% at MOI 5, p<0.0001) than hypoxia(27±5.52% at MOI 0.5, 17±2.18% at MOI 5, p<0.001), and cell death wasfound to be significant at MOI 5 in both normoxic and hypoxic conditions(29±1.37% and 22±5.82% respectively, p<0.0001) (FIG. 8b ).

Effects of HSV1716 Infection on Human Macrophages HSV1716 EffectivelyKills Human Macrophages

To determine if macrophages could be used as a delivery system forHSV1716 therapy, it was fundamental to test the consequences of viralinfection on macrophage cells. After 3 days from isolation, MDM wereharvested and counted; 1×10⁶ cells were seeded in 6-well plates. Oncecells attached to the plastic, infection was performed. HSV1716 wasadded at MOI 0 (control), 5 and 50. Cells were incubated in normoxic andhypoxic conditions for 96 hours. Medium was replaced with fresh mediumafter 24 hours, and on day 4 supernatant was collected from each wellfor further studies. After 96 hours, plates were harvested and cellviability was analysed by flow cytometry. Results showed an effectivekilling of cells at both MOI 5 and 50, with high percentages of celldeath under both normoxic conditions (63±2.76% at MOI 5, 62±2.42% at MOI50, p<0.0001) and hypoxic conditions (43±4.91% at MOI 5, p<0.001, and57±7.34% at MOI 50, p<0.0001) (FIGS. 9, 10).

Infected Macrophages Release Viral Particles

Since more than half of cells are killed by HSV1716, the considerableviral replication following infection and lysis of cells should lead tothe release of viral particles in the microenvironment. Therefore, toconfirm the ability of HSV1716 to kill and replicate in macrophages,supernatant from each well was collected after 96 hours from infectionand analysed. Samples, consisting of medium acquired from cells infectedat M010, 5 and 50 in both normoxic and hypoxic conditions and thepresence of viral particles in the supernatant was determined bytitration. HSV1716 was detected in the supernatant of MDM infected atMOI 5 and 50, with higher concentration for the higher MOI, while novirus was observed in control groups (Table 1 (FIG. 11)). Interestingly,samples collected from cells infected under hypoxic conditions showed a2.5-fold greater concentration (at MOI 5) and a 4-fold greaterconcentration (at MOI 50) of HSV1716 compared to their normoxicequivalents (FIG. 12).

To reaffirm this result, infection of tumor cells with conditionedmedium collected from infected MDM was performed. LNCaP cells wereseeded into 12-well plates (1×10⁴ cells/well) and infected with 100 μlof MDM supernatant in 1 ml RPMI. After 120 hours from infection, cellswere harvested and analysed by flow cytometry. After PI staining,however, cell death was found to be only significant at MOI 50, in cellsinfected with conditioned medium collected from MDM under normoxicconditions (40±7.26%, p<0.01), in contrast with what was expected fromtitration studies (higher cell death under hypoxic conditions) (FIG. 8).

HSV1716 Infection Modifies Macrophage Gene Expression

To investigate how infection with HSV1716 changes MDM gene expression,mRNA levels of cytokines and growth factors of interests were quantifiedusing quantitative RT-PCR. 1.5×10⁶ cells were seeded into 6-well platesand infected at MOI 50; plates were incubated under normoxic and hypoxicconditions, to understand possible alterations in gene expression due tothe different environment. 48 hours post infection, cells were harvestedand mRNA was extracted from infected cells and control groups. cDNA wasthen synthesised from RNA, and plated into 384-well plates with primersof genes of interest: the pro-inflammatory cytokines IL-6, IL-8, TNF-α,IL-1, CXCL1; the transcription factor NFκB, the anti-inflammatorycytokines IL-10 and CXCL-6, the growth factors VEGF-A and TGF-β. β-actinwas chosen as constitutively expressed housekeeping gene. Afterperforming RT-PCR, mRNA levels of each gene were normalised for β-actinconcentration and fold changes in the expression were calculated. Geneinduction profile caused by HSV1716 infection was obtained in duplicate.Fold changes in expression calculated for each gene were reported;results suggest that, at MOI 50, HSV1716 is an inducer ofpro-inflammatory cytokines (higher induction profile of IL-8, IL-1 andthe pro-inflammatory transcription factor NFκB in hypoxic conditions).At the same time, expression of NFκB and the anti-inflammatory TGF-β andIL-10 were reduced in normoxia (despite an apparent induction of thelatter under hypoxic conditions), while no detectable alteration of geneexpression was observed for the chemokines CXCL-1 and CXCL-6.Interestingly, a markedly high induction of VEGF-A after infection wasobserved under hypoxic conditions (Table 2 (FIG. 14)).

Infiltration of spheroids with MDM leads to tumor shrinkage

To investigate if delivery of HSV1716 to tumors and, specifically, tothe hypoxic core can be mediated by the use of macrophages, tumorspheroids were generated. The use of spheroids has the advantage,compared to 2D cultures, of being constituted by an oxygen-depletedcentral area surrounded by a well-oxygenated zone; therefore, a spheroidmimics 3D tumors. Tumor spheroids were generated on day 1 using LNCaPcells (1.5×10⁴ cells seeded into 2% agarose-coated 96-well plates). 3days after plating cells (day 4), spheroids of 800 μm/1 mm diameter haddeveloped. MDM were infected with HSV1716 at MOI 50 on day 3 andincubated for 24 hours, non-infected cells were used as controls. On day4, cells were harvested and counted; spheroids were infiltrated with5×10³ MDM, both at MOI 0 (control MDM) and MOI 50 (infected MDM). Inaddition, control spheroids (non-infiltrated) were taken into account.Plates were incubated for further 120 hours (until day 9). On day 6,after 72 hours from MDM infection, pictures were taken using afluorescence microscope, to visualise the presence of HSV1716 (labelledwith GFP) inside the spheroids. Images revealed the presence ofHSV1716-infected MDM; however, MDM seemed to be confined to the viablerim which surrounds the hypoxic core, while no GFP(+) cells wereobserved in the inner areas of spheroids. Central areas of spheroids(control groups) were found to be slightly necrotic and markedly darkerthan what was expected. On day 9, spheroids were further observed underthe microscope and pictures were taken; no significant alterations inthe shape and size of spheroids were detected. Spheroids were harvestedand washed after 5 days from infiltration (day 9) and cell viability wasanalysed by flow cytometry; while no significant cell death was observedin control groups and in spheroids infiltrated with non-infected MDM,infiltration with MDM infected at MOI 50 caused oncolysis of 51±5.92% ofcells (p-value <0.01) (FIG. 15).

Discussion

The findings revealed that prostate cancer cell lines LNCaP and PC3 andbreast cancer cell line T47D are sensitive to HSV1716, indicating thatthis virus could be used as a therapy to treat a broad range of tumors.Although at different extent, both prostate and breast cancer cell lineswere responsive to HSV1716 infection at MOI 5 after 72 hours (LNCaP,PC3) and 120 hours (T47D); in addition, the detection of high levels ofvirus uptake in living cells suggests that further cytotoxic effectcould be induced over time. Percentages of cell death were similar innormoxic and hypoxic conditions for all the cell lines tested (nostatistical significance was observed between the two groups): thisresult indicates that hypoxia does not confer resistance to HSV1716 totumor cells in vitro; HSV1716, therefore, could potentially be used tokill difficult to treat hypoxic areas of cancer. Interestingly, despitethe fact that significant cell death is observed under oxygen-depletedconditions, virus uptake in hypoxia is generally lower than the uptakein normoxia for both prostate and breast cancer cell lines—and is notsignificant for PC3. However, the high levels of cell death reportedsuggest a greater sensitivity to HSV1716 in hypoxic conditions.

The studies carried out on human macrophages showed that they aresensitive to HSV1716. The ability of HSV1716 to kill macrophages after96 hours from infection has a fundamental importance, as it implies thepossibility of exploiting MDM as a delivery system for the virus, whichwill be transported inside the hypoxic areas of tumors by MDM,replicate, lyse MDM and, subsequently, infect and kill the nearby cancercells. To further demonstrate that HSV1716 replication in MDM leads tothe release of viral particles in the microenvironment, supernatant wascollected from infected macrophages, with the aim of analysing it anddetecting the presence of HSV1716. Results revealed that the viralconcentration increases with an increasing MOI, as expected;interestingly, under hypoxic conditions replication and release of viralparticles were 3 fold greater than in normoxia. When LNCaPs wereinfected with the same supernatant, however, after 120 hours cell deathwas only significant in normoxia, at M0150, whereas no significantvalues were observed under hypoxic conditions or at lower MOI. Thisresult could be explained by the fact that the amount of viral particlesobserved after titration of supernatant, although higher in hypoxia, isgenerally not greater than 3×10³ PFU/ml: such quantity could be notsufficient for the virus to kill cells, considering that 100 μl ofsupernatant containing HSV1716 at 3×10³ PFU/ml (or less, in case of MOI5) were used to infect 2×10⁴ cells; this means that LNCaPs were infectedat MOI <0.05—an extremely low MOI (bearing in mind that, when performinginfection with HSV1716, significant cell death was only observed at MOI5). However, such low values of HSV1716 detected in MDM-conditionedmedia implies release of the virus, and the importance of this findingis clear when considering that, in a putative therapeutic approach, oncedelivered through MDM and released in the environment, viral particleswould encounter tumor cells, infect them, replicate, further amplifyingthe amount of viral copies, and subsequently disseminate widely intotumors.

To test the ability of MDM to deliver HSV1716 to tumors, andspecifically to hypoxic areas, multicellular 3D spheroids were prepared,with the aim of mimicking the structure of a real tumor. The mainobjective was to understand if MDM actually deliver the virus,sufficiently for cell death to be induced in 3D spheroids. Therelatively large diameter of spheroids (800 μm-1 mm) allowed theobservation of possible alterations in shape and size under themicroscope. In addition, the presence of GFP-labelled HSV1716 gave theopportunity to detect the presence of infected MDM inside the spheroidsand, therefore, to observe if MDM actually reached the hypoxic core.

Significant cell death was observed in spheroids infiltrated withinfected MDM (MOI 50) compared to control groups (p-value =0.009) andspheroids infiltrated with non-infected MDM (p-value =0.004).

HSV1716GFP was observed in spheroids treated with infected MDM, after 72hours from MDM infection (day 6) with weak gfp fluorescenceco-localising with the oxygenated rim. The absence of pronounced greenstains could be due to the small quantity of MDM used (only 5×10³ cellswere infiltrated with each spheroid). However, delivery of HSV1716 wassuccessful as spheroids infiltrated with infected MDM showed significantlevels of cell death (51±5.92%, p-value <0.01) suggesting that HSV1716replicates inside MDM and spreads in the micro-environment, ultimatelykilling tumor cells.

To understand how HSV1716 infection modifies gene expression in MDM,RT-PCR was performed. Genes of interest were selected based on theirimmune properties, the pro-inflammatory cytokines 1L-8, IL-6, TNF-α,CXCL-1, the anti-inflammatory cytokines IL-10, CXCL-6 and the factorsNFκB, VEGF-A, TGF-β. Virus infection of human cells generally leads toactivation of signalling pathways that cause the induction ofpro-inflammatory cytokines and transcription factors (Mogensen, T. H.,and S. R. Paludan, 2001 Molecular pathways in virus-induced cytokineproduction. Microbiology and Molecular Biology Reviews 65: 131-+). Itwas considered interesting, therefore, to analyse both the effect ofHSV1716 infection at MOI 50 on MDM gene expression and differencesbetween infections performed under normoxic and hypoxic conditions.

HSV1716 infection at MOI 50 caused the induction of pro-inflammatorycytokines by 48 hours, and the increase in expression was especiallyobserved under hypoxic conditions, whereas no considerable changes wereobserved in normoxia. Cytokines IL-8 and IL-1 were found to be 5- and7-fold upregulated, respectively, in hypoxia. A 5-fold increasedexpression under hypoxic conditions was also observed for NFκB; however,surprisingly, NFκB is down-regulated by 5 folds in normoxia. Thisfinding suggests a different response of MDM to HSV1716 which could havehigher inflammatory properties in the absence of oxygen. If this is thecase, this would suggest that HSV1716 acquires a greater viral potencyin hypoxia: this result would further support the rationale of usingvirus delivery by MDM to target central areas of tumor, difficult toaccess through different ways.

The anti-inflammatory cytokine IL-10 and the growth factor TGF-β aredown-regulated by 5 folds after HSV1716 infection, but only in normoxicconditions. Indeed, under hypoxia, a 4-fold increase in theanti-inflammatory IL-10 expression was observed, possibly opposing thepro-inflammatory effect. Interestingly, there is strong up-regulation ofVEGF-A under hypoxic conditions (21 fold), which could be partly due bythe fact that VEGF-A is normally involved in the hypoxic response.

In summary, this study demonstrates that HSV1716 induces tumor celldeath in prostate and breast cancer cell lines, and is able to replicatein MDM and disseminate in the surrounding microenvironment. In addition,results show that when delivered through MDM, HSV1716 causes cell deathin multicellular 3D spheroids; therefore, the macrophage-mediateddelivery of oncolytic HSV1716 to tumors constitutes a possibletherapeutic approach to treat solid tumors. The great safety profile ofHSV1716, shown by previously performed clinical trials, makes thepossibility of using it as a MDM deliverable therapy an excitingopportunity to further increase the range of treatments that can beoffered to cancer patients.

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Lan, C. Y., X. Huang, S. X. Lin, H. Q. Huang, Q. C. Cai et al., 2013Expression of M2-Polarized Macrophages is Associated with Poor Prognosisfor Advanced Epithelial Ovarian Cancer. Technology in Cancer Research &Treatment 12: 259-267.

Lewis, J., R. J. Landers, R. D. Leek, K. Corke, A. L. Harris et al.,1997 Role of macrophages in tumor angiogenesis: Regulation by hypoxia.Journal of Pathology 182: A1-A1.

Lewis, J. S., R. J. Landers, J. C. E. Underwood, A. L. Harris and C. E.Lewis, 2000 Expression of vascular endothelial growth factor bymacrophages is up-regulated in poorly vascularized areas of breastcarcinomas. Journal of Pathology 192: 150-158.

Li, X., H. Kimura, K. Hirota, H. Sugimoto and H. Yoshida, 2005 Hypoxiareduces constitutive and TNF-alpha-induced expression of monocytechemoattractant protein-1 in human proximal renal tubular cells.Biochemical and Biophysical Research Communications 335: 1026-1034.

MacKie, R. M., B. Stewart and S. M. Brown, 2001 Intralesional injectionof herpes simplex virus 1716 in metastatic melanoma. Lancet 357:525-526.

Martinez, F. O., S. Gordon, M. Locati and A. Mantovani, 2006Transcriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization: New molecules and patterns of geneexpression. Journal of Immunology 177: 7303-7311.

Mogensen, T. H., and S. R. Paludan, 2001 Molecular pathways invirus-induced cytokine production. Microbiology and Molecular BiologyReviews 65: 131-+.

Mukhtar, R. A., A. P. Moore, V. J. Tandon, O. Nseyo, P. Twomey et al.,2012 Elevated Levels of Proliferating and Recently MigratedTumor-associated Macrophages Confer Increased Aggressiveness and WorseOutcomes in Breast Cancer. Annals of Surgical Oncology 19: 3979-3986.

Example 3 Isolation and Culture of Human Macrophages

Mononuclear cells were isolated from platelet-depleted buffy coats(Blood Transfusion Service, Sheffield, UK) using Ficoll-Paque Plus(Amersham Pharmacia, St. Albans, UK) and monocyte-derived macrophages(MDM) prepared as described previously^(21,22).

Endothelial Cell Cultures

Human Umbilical Vein Endothelial Cells (HUVEC) were seeded for 24 h ontocollagen-coated (0.1 mg/ml, human type IV) membranes containing a 5 μMpore PET membrane (Neuroprobe).

Human Multi-Cellular Tumor Spheroids

Human prostate cancer cell line, LNCaP, were seeded (5×10³) in 100 ulmedium into each well of a 2% agarose (Sigma, Dorset, UK) coated 96-welltissue culture plate. After 7-10 days, each well contained a tumorspheroid with an average diameter of 700-800 um²¹.

Infection of Primary Macrophages

Day 3 MDMs were infected with a replication deficient adenovirus(CMV-AdV5-GFP (driven by a CMV promoter). Virus optimization and GFPexpression levels are described in²¹.

Cellular Uptake of Magnetic Nanoparticles by Macrophages

MDMs (infected with AdCMV-GFP) were cultured overnight with 100 ug/mlSPIOs (25 nm) (Sigma-Aldrich, Poole, UK). SPIO accumulation in cells waspreviously assessed by flow cytometry and confirmed by attraction of thecells towards a magnet placed at the side of the culture dish asobserved by light microscopy as described in Muthana, M. et al. A novelmagnetic approach to enhance the efficacy of cell-based gene therapies.Gene Ther (2008). Cell viability following SPIO uptake by macrophageswas also measured and compared to cells that were not incubated withSPIOs using the DNA dye propidium iodide (PI). No statisticallysignificant difference was observed between the two groups p=0.4 (FIG.20c ) N=3.

In Vitro Trans-Endothelial Flow Assay

The trans-endothelial migration (TEM) chamber was assembled as shown in(FIG. 20a ). SPIO-loaded MDM (1.5 ×10⁵ cells/mi in PBS+2% FCS) wereallowed to flow over the HUVEC monolayer at typical venous flow rates(1.1885 ml/min) at a sheer stress of 1.4 Dynes/cm², this is equivalentto blood flow through post-capillary venules. The TEM chamber waspositioned directly in the iso-centre at ˜5 mm distal of a 7 Teslamagnet (Bruker BioSpecAVANCEII, 310 mm bore, MRI system B/C 70/30). Theflow in the chamber was in the -z direction (in and out of the magnetbore). We used pulsed gradients 2 ms on, 7 ms off as described byReigler et.al¹³. To steer SPIOs into the chamber containing tumorspheroids we applied a pulsed -y gradient at 50% strength to avoidover-heating (˜300 mT/m) for 30 minutes. Post MRT a ¹H volume resonator(Bruker, 300 MHz, 1 kW max, outer diameter 118 mm/inner diameter 72 mm)allowed capture of MR images (FLASH and RARE).

Spheroid infiltration by MDMs was then assessed using a fluorescentmicroscope to detect the GFP positive cells and flow cytometry usingenzymatically-dispersed spheroids. To determine the iron content withinSPIO-loaded macrophages, cell pellets were solubilized in 70% nitricacid for 7-14 days prior to analysis. Iron concentrations werequantified against a calibration standard iron solution (FischerScientific, Loughborough, UK) by Atomic Emission Spectroscopy usingVarian Vista-M PX¹⁴.

Orthotopic Prostate Xenograft Model

Male CD1 athymic mice were used in these studies (Charles Rivers, UK).One million LNCaP:LUC cells (a kind gift from Professor Magnus Essand,Uppsala Sweden) were mixed 1:1 in Matrigel and injected into thedorsolateral prostate. Tumor size was determined by assessment usingbioluminescent IVIS imaging and measuring the daily weights of the miceas described in in Muthana, M. et al. Macrophage Delivery of anOncolytic Virus Abolishes Tumor Regrowth and Metastasis AfterChemotherapy or Irradiation. Cancer Res, doi:0008-5472.CAN-12-3056[pii]10.1158/0008-5472.CAN-12-3056 (2013). Tumor-bearing mice were usedin experiments approximately 14 days following implantation or 21 daysin the metastases model when the pulmonary tumors develop followingimplantation of the tumor cells into the prostate²¹.

Use of the MRI Scanner to Direct Cell Movement

Three million MDMs with or without SPIOs were administered via tail veinin 100 μl volume of PBS (n=5), control groups received 100 ul PBS (n=5),or 100 ul PBS containing 3×10⁶ macrophages without SPIOs (n=5).Immediately after MDM administration mice were anaesthetized withgaseous isoflurane and then secured within a magnet-compatible holdingcapsule and MR targeting was carried out immediately.

Mice were split into 2 groups of n=5. Group 1 was a time-matched controlwithout MR targeting and Group 2 underwent 1 hour of MR targeting (seeabove) with gradients selected for coarse steering to the tumor site forthe Prostate (-z, -y). For steering to the lungs (+z and −y gradients),the absence of an x gradient should ensure even distribution of magneticparticles in each lung.

The force on magnetically labeled cells is dependent on whether theSPIOs have become magnetically saturated. When unsaturated, the force isdependent on the magnetic susceptibility of the SPIOs, the magneticfield and also the magnetic field gradient (Pankhurst, Q. A., Connolly,J., Jones, S. K. & Dobson, J. Applications of magnetic 443″nanoparticles in biomedicine. J Phys D Appl Phys 36, R167-R181,(2003)).

However once the SPIOs reach saturation, the force is no longerdependent on the magnetic susceptibility of the particle but thesaturation magnetization and as such only the magnetic field gradientwill affect the force applied to the cells (Riegler, J. et al. Targetedmagnetic delivery and tracking of cells using a magnetic resonanceimaging system. Biomaterials 31, 5366-5371, (2010).) SPIOs typicallyreach magnetic saturation well below 1 T, for example in Riegler et al.2013, where the SPIOs become saturated at around 300 mT, therefore MRTis feasible on clinical MRI systems provided the same magnetic fieldgradient is used ˜300 mT/m.

Following MRI-steering, high-resolution RARE and FLASH images of thetumor (prostate only) were taken. Once complete relaxometry-using MSEand MGE was performed to assess the transverse relaxation rates. Aftertreatment, animals were sacrificed and tissues including tumors, kidney,liver, lungs and spleen, were either paraffin wax embedded and fixed forimmunohistochemistry or analyzed by flow cytometry to determinemacrophage uptake.

Endothelial Cell Cultures

Human Umbilical Vein Endothelial Cells (HUVEC) were obtained fromPromocell, (Heidelberg, Germany) and used in the experiments up topassage 8. Cells (150,000) were seeded for 24 h onto collagen-coated(0.1 mg/ml, human type IV) membranes containing a 5 μM pore PET membrane(Neuroprobe). This resulted in a confluent monolayer of HUVECs onfilters as seen by CD31 staining (data not shown).

Infection of Primary Macrophages

Day 3 MDMs were infected with a replication deficient adenovirus(CMV-AdV5-GFP). The E1A/B-deleted adenoviral vectors, CMV-AdV5-GFP(driven by a CMV promoter) was isolated from a single plaque, expandedin 293 human embryonic kidney (HEK) cells All the viruses were purifiedby double caesium gradient centrifugation, and titered by plaque assayon 293 cells with the titer expressed as plaque forming units(PFU)/cell. Virus optimization and GFP expression levels in macrophagesare described in²¹.

Flow Cytometric Analysis

Single cell suspensions were obtained by trypsinizing MDMs (includingco-transduced MDMs). Cells were then incubated for 30 min at 4° C. withmouse anti-CD14, 1:100 in PBS containing 1% BSA (Sigma) to preventnon-specific antibody binding. Alternatively, spheroids were digestedusing 0.25% trypsin/EDTA to separate the tumor cells and infiltratedmacrophages and cell death was analysed by flow cytometry by addingpropidium iodide (Sigma) to the cells immediately before running on theflow cytometer.

Cellular Uptake of Magnetic Nanoparticles by Macrophages

For the nanoparticle cellular uptake studies, MDMs (infected withAdCMV-GFP) were cultured overnight with magnetic nanoparticles 100 μg/mlSPIOs (25 nm) (Sigma-Aldrich, Poole, UK). MNP accumulation in cellsfollowing incubation with SPIOs was assessed by flow cytometry, thisincluded measuring cell viability with propidium iodide (PI) asdescribed by us¹⁴ and confirmed by attraction of the cells towards amagnet placed at the side of the culture dish as observed by lightmicroscopy (Leica Microsystems UK Ltd).

HSV1716 Virotherapy

For therapeutic studies LNCaPs or macrophages were infected withHSV1716GFP (an HSV1716 variant with a GFP expression cassette insertedin the deleted ICP34.5 loci) at a multiplicity of infection (MOI) of 5or 50. Cell death was assessed by flow cytometry 96 h post infectionusing PI staining. Viral particles were detected in clarifiedsupernatants of infected macrophages using a titration assay on Verocells to determine plaque-forming units.

Mice received tail vein injections of either 3 million MDM alone orarmed with HSV1716 at MOI 50, 1×10⁷ pfu HSV1716 only or PBS (n=5mice/group). Of note, 3 groups of mice were administered MDM+OV, onegroup underwent MRT for 1 h, one sat in the MRI scanner for 1 h but hadno MRT (MDM+OV no MRT) and another group did not enter the MRI scanner(MDM+OV). Tumor size was monitored by IVIS Lumina II imaging (IVIS,Caliper Life Sciences). Animals were sacrificed once tumors reached themaximum volume permitted by UK Home Office Regulations, and 1 hourbefore sacrifice, mice were injected intravenously FITC:Lectin (used fordetecting tumor vasculature). Of note, mice receiving PBS and MDM onlywere culled on day 14-post treatment due to large tumor size. All othertumors were removed on day 21. Excised tissues including tumors, kidney,liver, lungs, and spleen were embedded in OCT or paraffin wax forhistologic labeling studies.

Analysis

Tissues were divided into two; one part was formalin fixed forimmunohistological analysis and the other was dissected free of adherentfibrous and fatty tissue and treated with collagenase.

Flow cytometry: cell viability was determined using LIVE/DEAD FixableViolet Dead Cell Stain Kit (Invitrogen). All FACS data were analyzed onan LSR II flow cytometer (BD Biosciences) using FlowJo software (TreeStar).

Histology: Five micron sections of all organs were incubated withspecific antibodies for target antigens; for the vasculature we usedCD31 (1:100), (AbD Serotec) and for macrophages human CD68 (Dako, Ely,UK) at 1:100 and to detect adenovirus we used E1A at 1:50 (Millipore,UK). A biotinylated secondary antibody system was used in conjunctionwith a streptavidin-conjugated HRP. Peroxidase activity was localisedwith diaminobenzidine (Vectastain Elite ABC kit, Vector Labs). To detectiron in the tumors (where cell densities were high) sections werestained with Perls Prussian blue and counter-stained with eosin forimproved contrast. To detect cancer cells in the lungs all lung sectionswere stained with Epithelial cell adhesion molecule (EPCAM) orHematoxylin and eosin (H&E). All immune-localization experiments wererepeated on multiple tissue sections and included isotype-matchedcontrols for determination of background staining.

Statistical Analysis

Data are means±SEM. Student's t test were used to analyze thestatistical significance of the data. Differences were termedsignificant a P value of less than 0.05.

Supplementary Methods

Mouse procedures and human monocyte isolation were conducted inaccordance with the University of Sheffield Ethics Committee and UK HomeOffice Regulations under the Animals (Scientific Procedures) Act 1986.

Results

We show that therapeutic cells armed with an oncolytic virus (HSV1716)can be magnetically labeled using super-paramagnetic iron oxidenanoparticles (SPIOs) and then steered from the bloodstream into deeptarget tissues (primary and secondary tumors) using pulsedmagnetic-field gradients within a magnetic resonance imaging (MRI)system. Use of this technique resulted in a marked increase in celldelivery to tumors and a significant reduction in tumor burden andmetastasis. Our study, therefore, shows that clinical MRI scanners couldbe used, not only to image such magnetically labelled cells after theirinjection into the body, but also to steer them specifically to one ormore target sites within the body. We describe the use of magneticresonance targeting (MRT) to increase delivery of macrophages to tumors.

We show that it is possible to manipulate the spatial field gradientcoils of the MRI scanner to shape the magnetic field in/around a tumor,thereby non-invasively steering magnetically labeled cells towards it(FIG. 16).

We previously showed that such MRT could be used to both image and movecells in an in vitro vascular bifurcation model (a 2D tube that mimicsarterial bifurcation)¹³. Here, we show that MRT can also be used to‘steer’ magnetic macrophages in vivo—i.e. from the bloodstream into twotarget organs, orthotopic prostate tumors and their pulmonary metastasisin mice. We have used macrophages as an example of a cellular vehicle asthese cells are highly phagocytic allowing them to readily consume SPIOswhilst retaining their magnetic properties^(14,18,19). Such bonemarrow-derived cells are increasingly being used in cell-based therapiesfor such diseases as cancer²⁰⁻²², infarcted myocardium²³, spinal cordinjury²⁴, cerebral ischemia²⁵, degenerative diseases like Parkinson'sDisease²⁶ and Alzheimer's Disease²⁷.

Before applying MRT techniques in vivo we first established that apre-clinical 7 T MRI system fitted with a 600 mT/m gradient coil setcould generate substantial actuation forces on magnetic macrophages invitro by steering them across an endothelial layer into 3D human tumorspheroids (MTS). To do this, we designed a trans-endothelial migration(TEM) flow chamber in which human macrophages circulated across thesurface of a perforated membrane coated with a layer of human vascularendothelial cells, thereby mimicking flow in tumor venules. MTS werecultured in a non-adherent chamber below the membrane (FIG. 20a ). Humanmacrophages transfected to express a GFP reporter adenovirus(Ad-CMV-GFP) were loaded with SPIOs (1.18 ug/ml±0.3)¹⁴ and then steeredacross the membrane into MTS when the chamber was placed in theiso-centre of a high-field (7 T) pre-clinical MRI system.

MRT experiments used a pulsed magnetic field gradient (2 ms on, 7 msoff, 50% strength ˜300 mT/m¹³) for 1 hour in the direction of thespheroids (FIG. 20a ) with an effective additional magnetic fieldoffset, B_(off)˜+1.5 mT around the MTS site. In control conditionssamples were exposed to the magnetic field of the scanner but gradientswere not pulsed. Using MRT, we found a T₂*-weighted signal lossindicating higher concentration of iron in comparison to the controlsamples for MRT exposed samples (n=6) (FIG. 20 ci) and GFP-expressingmacrophages were clearly visible within MTS (FIG. 20 cii). Flow analysisfurther confirmed macrophage uptake with significantly (P=0.0001) moreviable infiltrating CD14⁺/PI⁻ expressing macrophages with MRT(29.7%±2.6) than without (2.9%±1.8) (FIG. 20 c iii-iv).

We then investigated whether such an MRI gradient system could be usedto steer magnetic macrophages to tumors in vivo (FIG. 17). Three millionSPIO-loaded macrophages were administered intravenously to mice bearingorthotopic primary and metastatic (lung) prostate tumors. A pulsedmagnetic field gradient¹³ was applied for 1 hour, in the direction ofthe prostate (FIG. 17a ), with an effective magnetic field offset,B_(off)˜+7 mT on top of the static magnetic field of the scanner (B₀=7T). The control group were exposed to the static magnetic field of thescanner in the absence of the steering gradients (no MRT).

MRT significantly (p=0.0001) increased uptake of SPIO-loaded macrophagesin primary prostate tumors (42.2%±2.5) compared to the control group(7.17%±0.8) (FIG. 17b ). Moreover, these cells were present throughouttumors, with very few signs of cell clumping in the tumor vasculaturefollowing MR targeting as seen by labeling sequential sections of tumorsusing an antibody against human CD68 (a pan macrophage marker) and ahistological stain for iron (Prussian Blue or ‘PB’) (FIG. 17c ). MRIsteering of macrophages did not adversely affect the tumor vasculature(FIG. 21a ) and in the multi-echo RARE MR images of tumors littledifference can be seen between the MRT and no MRT groups (FIG. 17d ).This is most likely due to the blood pool iron content per voxel.However, a marked difference between SPIO injected and non-injectedsubjects is evident in the T2-weighted long TE images, with loss insignal intensity within the tumor (distorted MRI image with MRT comparedto control) indicating the presence of high concentrations of iron (FIG.17e ). In an effort to assess the increased uptake of magneticmacrophages in vivo we used MR relaxometry to measure the MR transverserelaxation decay rate (R₂) in tumors in both groups. R₂ measurementswere 21.8s⁻¹ for the MRT group and 18.8s⁻¹ for the control group. NormalR₂ decay rate f tumor tissue without the presence of any SPIOs is alsoincluded for comparison (10.5s⁻¹). A higher decay rate indicatedincreased iron uptake for the MRT group—suggesting it is possible toassess the uptake with MRI, as seen with the post mortem analysis. Thesignificant difference in R₂ values was used to estimate the best echotime for analysing signal differences with spin echo-based MRI sequencesat a TE of 60 ms, here MRT leads to a 10% decrease in signal over thetime-matched controls.

Additional controls included tumor-bearing mice: (i) with unlabelledmacrophages and MR targeting, and (ii) with unlabelled macrophageswithout MR targeting. For these control groups, we detected very fewmacrophages within tumors as confirmed by MRI imaging (FIG. 21c ) andflow cytometry of enzymatically dispersed tumors (FIG. 21d ). Of note,we detected virtually no human CD68+macrophages in other tissuesincluding the liver (<2% of all cells/tissue section), spleen (<1%) andkidneys (none detected) (FIG. 22).

MRT has particular application when tumors are difficult or impossibleto remove surgically, as in the lung, brain, liver or spinal cord.Separate MRT sessions could enable targeting of a cell-based therapy toone or more metastatic lesions in cancer patients. In a second in vivoexperiment we steered magnetic macrophages into lungs containingmicro-metastases in our tumor-bearing mice. MRT was again used to steermagnetic macrophages towards the lungs following administration of 3million macrophages. Mice without application of MRT but exposed to themagnetic field of the scanner, for the same length of time were used asa time-matched control.

Flow cytometric analysis of enzymatically dispersed lungs showed thepresence of significantly more human CD14+ macrophages following MRTthan in the control group (17.7%±4 vs. 4.4%±2.6, respectively) (FIG. 18a). This was also confirmed by histological staining of lungs, whereCD68+ human macrophages were detected in or close to the metastaticdeposits within the lungs of mice following MRT (FIG. 18b and FIG. 23a). These macrophages were also positive for Prussian Blue (FIG. 18c )and their iron content was also visible following H&E staining (FIG. 23b). We inspected the morphology of CD31+ blood vessels in the lungsfollowing their uptake of SPIO-labelled macrophages with or without MRT(FIG. 18c ). In addition, we examined every blood vessel in each of the5 tumors in these 2 groups of mice and found no differences between thetwo groups. We could not see signs of endothelial cell disruption, norwere there any signs of blood clotting (e.g. platelet aggregation) in,or on the abluminal side of blood vessels after MRI targeting. Due tothe short T2/T2* of lung tissue it was not possible to image the lungparenchyma with conventional ¹H MRI techniques at high field for in vivovalidation of increased uptake. Future technical developments may makethis possible, for example the use of hyperpolarised gases in theairspaces could be used as an indirect MR signal detection method²⁸.Nevertheless, in different organs or soft tissues, or on clinicalsystems, T2* imaging may have a place.”

In a final experiment to assess the therapeutic benefits of MRT wetargeted SPIO-loaded macrophages armed with the therapeutic oncolyticvirus (OV) HSV1716 to tumor bearing mice. HSV1716 replication issupported by PC3 prostate cancer cells {Conner and Braidwood, CancerGene Ther. 2012 July; 19(7):499-507} and here we show for the first timeoncolysis in LNCaP cells in both hypoxic (0.5% O₂) and normoxic (20% O₂)conditions (FIG. 24a ). HSV1716 is readily taken up by macrophages andwhilst uptake is significantly higher (p=0.002 at MO15 and p=0.001 atMO150) in normoxic culture conditions (FIG. 24b ), viral replication isgreater in hypoxia and macrophage cell death is equally effective in ahypoxic environment (FIG. 24c,d ). In our in vivo model, tumor bearingmice received either a single intravenous injection of OV-carryingmacrophages (MDM+OV) but were either not exposed to the MRI scanner,were static in the scanner but without MRT (MDM+OV (no MRT)), or wereexposed to the scanner with MRT (MDM+OV+MRT). For the purpose ofcomparison “free” OV was administered to a separate group of mice.Additional control groups of mice received either 100 ul salinetreatment (Control) or 3 million macrophages (MDM) intravenously. OV(1×10⁷ pfu) {Sorensen et al., J Nucl Med 2012 53:647-654} alonesignificantly (P<0.03) delayed primary tumor growth for up to 7 dayscompared to mice receiving PBS or MDM only (FIG. 19a ). This effect wassignificantly prolonged with macrophage-mediated delivery of HSV1716(p<0.006 at day 14 and p<0.007 day 21). Of note, no differences wereobserved in mice receiving MDM+OV and MDM+OV (no MRT) where the latteris exposed to the scanner but with no steering. However, MRT targetingof our macrophage therapy was not only superior in reducing the size ofthe primary tumors from day 7 onwards this also delayed primary tumorregrowth for the entirety of the experiment (FIG. 19a ).

Bioluminescence of mice receiving macrophage OV therapy with or withoutMR targeting on the first day of treatment (day 0) and at the end of theexperiment (day 21) showed this marked reduction of the primary tumor(FIGS. 19a & b). This was confirmed visually on the MRI scans (FIG. 19c). Furthermore, tumors undergoing MR targeting followingmacrophage-delivered OV were significantly more necrotic (p<0.001) thanthose not receiving MR targeting (FIG. 19e ).

MR images of mice receiving macrophage OV therapy with or without MRT onthe first day of treatment (day 0) and at the end of the experiment (day21) reflect this marked reduction of the primary tumor. Interestingly,the tumors from mice treated with OV or MDM carrying OV wereconsiderably paler and less vascularized and this correlated with areduced microvessel density (MVD) compared with the PBS or MDM alonegroup. In mice undergoing MRT following macrophage-delivered OVsignificantly more necrosis (p<0.001) in tumors was observed than in theabsence of MRT.

We next determined how these therapies influenced the development ofpulmonary metastases. Few metastases were detected in mice injected withPBS or MDM alone since primary tumors in these groups had to be removedby day 14 (due to their size). Therefore, it was not valid to comparemetastases in these control groups with the other experimental groups.However, the formation of lung metastases was markedly reduced when micereceived MRT following delivery of OV-bearing macrophages in comparisonto when no MRT was used (FIG. 19f 0.8±0.37 vs. 3.8±0.95 p<0.02).

In summary, we show that an MRI scanner can be used to non-invasivelysteer cells to both primary and secondary tumors within the body leadingto a significant improvement in therapeutic outcome. Moreover,relaxometry measurements suggest that MRI post MRT can be used to assessthe efficacy of this approach. Whilst this study has focused on celldelivery to tumors, the technology could be used to target any cells(e.g. stem cells) to a given tissue including non-phagocytic cell typeswhich could be ‘magnetised’ using SPIO-conjugated antibodies directedagainst proteins on their cell surface.

The use of magnetic resonance targeting, which exploits the magneticfield gradients within magnetic resonance imaging systems to increasedelivery of cells, is ideally suited to deep or superficial tissue. Thequestion of clinical translation is dependent on the ability to providethe same targeting force on a clinical MRI system. Clinical scanners,with high performance magnetic field gradient systems of 300 mT/m, arealready in use and as such have the potential to produce similar forces.Moreover, we were able to image the cell distributions following MRT,indicating the possibility for real-time image-guided targeting using anMRI system. These findings support the potential value of MRT withconcomitant imaging for improved targeting of cells for therapy.

References for Example 3

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1-19. (canceled)
 20. An isolated ex vivo cell productively infected withan oncolytic herpes simplex virus wherein the cell is a macrophage andwherein the oncolytic herpes simplex virus is HSV1716 or a mutantthereof, wherein the oncolytic herpes simplex virus mutant is an ICP34.5null mutant.
 21. The cell of claim 20, further comprisingsuper-paramagnetic iron oxide nanoparticles.
 22. A method of treating adisease in a subject in need of treatment, the method comprisingadministering to said subject a preparation comprising a population ofoncolytic herpes simplex virus-infected cells of claim
 20. 23. Themethod of claim 22, wherein the disease is cancer.
 24. The method ofclaim 22, wherein the population of oncolytic herpes simplexvirus-infected cells further comprises super paramagnetic iron oxidenanoparticles.
 25. The method of claim 24, wherein the method furthercomprises applying a magnetic field to the subject in order to directthe population of cells of the administered preparation to a desiredlocation in the subject's body.
 26. The method of claim 25, wherein thedesired location is the site of a tumor.
 27. The method of claim 26,wherein the tumor is in an organ selected from the group consisting ofthe adrenal gland, adrenal medulla, anus, appendix, bladder, bone, bonemarrow, brain, breast, cecum, central nervous system, brain, cerebellum,cervix, colon, duodenum, endometrium, gallbladder, esophagus, heart,ileum, intestines, jejunum, kidney(s), lacrimal gland, larynx, liver,lung(s), lymph, lymph node, mediastinum, mesentery, myometrium,nasopharynx, omentum, ovary, pancreas, parotid gland, peripheral nervoussystem, peritoneum, pleura, prostate, rectum, salivary gland, colon,small intestine, spleen, stomach, testis, thymus, thyroid gland, anduterus.
 28. The method of claim 22, wherein the population of oncolyticherpes simplex virus-infected cells is prepared by contacting apopulation of cells comprising macrophages with a quantity of oncolyticherpes simplex virus under suitable conditions and for sufficient timeto permit productive infection of the macrophages.
 29. The method ofclaim 28, wherein the cells are maintained in culture under conditionsin which the virus is able to induce cell death.
 30. The method of claim29, wherein the population of oncolytic herpes simplex virus-infectedcells comprises a mixture of intact and lysed macrophages.
 31. Themethod of claim 29, wherein 1-50% of the population of oncolytic herpessimplex virus-infected cells of the preparation are dying or dead.
 32. Apopulation of oncolytic herpes simplex virus-infected cells of claim 20,wherein the population comprises a mixture of intact and lysedmacrophages.
 33. A population of oncolytic herpes simplex virus-infectedcells of claim 32, wherein 1-50% of the population of oncolytic herpessimplex virus-infected cells of the preparation are dying or dead.